Seasonal Changes in Free-Living Amoeba Species in the Root Canopy of Zygophyllum dumosum in the Negev Desert, Israel

The influence of seasonality and Zygophyllum dumosum root canopy on the species diversity of free-living amoebae at two soil depths (0–10 and 10–20 cm) was studied in a Negev Desert ecosystem in Israel. Free-living amoebae were extracted and identified after cultivation in non-nutritive agar plates. A total of 90 amoeba species were identified in the soil during the study period, with the most common genera present being Hartmannella, Platyamoeba, Vahlkampfia, Acanthamoeba, and Echinamoeba. Differences between the control soil and the soil under Z. dumosum were found mainly during the dry seasons, when 97% similarity was found between the two soil layers, which could be due to the effect of the shrub on the soil microenvironment. The amoeba community exhibited more species diversity in spring (reaching a value of 34 species) than in the winter (18 species) or summer and autumn (20 species), since the community has a time lag for becoming stabilized after the dry summer and autumn. This is one of the first studies on the amoeba population in the Negev Desert and elucidates the importance and the need for taking trophic and functional groups into consideration in order to understand biomineralization processes.     

Taxonomic Criteria for Limax Amoebae,with Descriptions of 3 New Species of Hartmannella and 3 of Vahlkampfia*

SYNOPSIS. Seven species of limax amoebae were isolated into clonal, monoxenic cultures with Aerobacter aerogenes from material collected from freshwater habitats. Studies were made of their trophic structure, nuclear division, cyst structure, some aspects of cytochemistry, and other characteristics. Six new species are described: Vahlkampfia inornata, V. avara, V. jugosa, Hartmannella limacoides, H. vermiformis, and H. exundans. The well-known species Naegleria gruberi (Schardinger, 1899) is re-described on the basis of 8 strains; its flagellated phase was found to be biflagellate, with rare exceptions. A correlation exists between the manner of locomotion and the pattern of nuclear division in the limax amoebae in the family Vahlkampfiidae and those in the genus Hartmannella. Trophic amoebae of all species had a PAS-positive surface layer, altho results with H. vermiformis and H. exundans were less definite than with other species. All species except H. limacoides formed cysts in culture. The cyst walls of all cyst-forming species were strongly PAS-positive, but results of the zinc chloroiodide test for cellulose were negative with the method used. The genus Hartmannella Alexeieff, 1912, is re-defined to include those species which assume a simple, monopodial limax-like form during locomotion and have nuclear division similar to that of metazoan cells and to distinguish it from the genus Acanthamoeba Volkonsky, 1931.     

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.     

Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Isolation and Molecular Characterization of Acanthamoeba Strains from Dental Units in Costa Rica

Free‐living amoebae are protozoa widely distributed in nature, which can be found in a variety of environments. Four genera are recognized as causal agents of infections in humans and animals: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. In this study, the presence of Acanthamoeba in dental units was determined and the isolates obtained were molecularly characterized; osmotolerance and thermotolerance assays were also performed to evaluate multiplication under these conditions, frequently associated with pathogenicity. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 14% of the units sampled. Osmotolerance and thermotolerance tests were positive for more than 80% of the isolates. Up to date, this is the first study that reports the detection, identification, and genotyping of Acanthamoeba isolated from dental units in Costa Rica and even in Latin‐America. Further assays to determine the potential pathogenicity of these Acanthamoeba isolates are underway.     

A systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype

Acanthamoeba is a genus of facultative human parasites that is currently classified into 17 genotypes (T1–T17) each of which arguably represents a species. These amoebae cause Acanthamoeba Keratitis (AK) a disease of the eye, and a rare but usually fatal Granulatomous Acanthamoeba Encephalitis (GAE). A database of strains derived from the literature and a number of fresh isolates has been constructed to detect trends of pathogenic and other associations with these genotypes. One genotype in particular, T4, was found to be over represented in human disease. The prevalence of this genotype has been commented upon previously, however T4 is also the most common type isolated from the environment. Our statistical analysis of the database allows us to claim that T4 is in fact the genotype most often associated with human disease, even after its abundance in the general environment is taken into account. T3 and T11 are closest relatives to T4 and they are the second and third most often associated with AK. A number of other more subtle correlations also emerge from this analysis.     

Pathogen–pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba

In this study, we compared the interactions of invasive and non-invasive strains of E. coli with clinical and environmental isolates of Acanthamoeba. The environmental isolate of Acanthamoeba exhibited significantly higher association with E. coli compared with the clinical isolates of Acanthamoeba. The ratio of E. coli per amoebae was more than 8-fold higher in the environmental isolate compared with the clinical isolates of Acanthamoeba. Interestingly, non-pathogenic environmental Acanthamoeba showed uptake and/or survival of the non-invasive E. coli. In contrast, clinical isolates of Acanthamoeba did not support uptake and/or survival of non-invasive E. coli. Using several mutants derived from K1, we demonstrated that outer membrane protein A (OmpA) and lipopolysaccharide (LPS) are crucial bacterial determinants responsible for E. coli K1 interactions and in the intracellular survival of E. coli in Acanthamoeba. The use of Acanthamoeba as a model to study E. coli K1 pathogenesis and to understand bacterial immune evasion strategies is discussed further.     

Isolation and identification of Acanthamoeba species related to amoebic encephalitis and nonpathogenic free‐living amoeba species from the rice field

Aims: Isolation and characterization of the clinically relevant amphizoic amoebas in vegetated farmlands, which may present a risk to farmers’ health. Methods and Results: Acanthamoeba species was isolated and characterized via morphological and molecular means in the rice field where the patient was exposed to rice paddy water which most probably was the point of infection. An Acanthamoeba sp. abundant in the rice field was identified. Genotyping showed the strain to be genotype T4, which was identical to the amoebic parasite found in patient’s cerebrospinal fluid. During the course of the study, three nonpathogenic free‐living amoeba species were also isolated and characterized for the first time in Taiwan. Conclusions: This study successfully located a possible source of granulomatous amoebic encephalitis in a patient and provided the first evidence that Acanthamoeba genotype T4 may be a potential pathogen in Taiwan. Significance and Impact of the Study: The integration of field survey, clinical data and morphological and genetic examination represents a sound strategy for investigation of the possible role of free‐living amoebae in causing human diseases. Future work should include investigating the potential contributory role of other nonpathogenic free‐living protozoa in disease of livestock or even human.     

Pathogenicity, Morphology, and Differentiation of Acanthamoeba

Acanthamoeba keratitis is sight threatening corneal infection caused by pathogenic Acanthamoeba. Previous studies have shown the genotypic differences between pathogenic and non-pathogenic species/strains of Acanthamoeba. In this study, we examined the morphological differences between pathogenic and non-pathogenic species/strains using scanning electron microscopy. Pathogenic Acanthamoeba exhibited higher number of acanthopodia (structures associated with the binding of amoeba to the target cells) as compared to non-pathogens. In addition, interactions of amoeba with the corneal epithelial cells were studied. Only pathogenic amoeba exhibited adhesion to epithelial cells. Further results indicated that phagocytosis occurs in the pathogenic amoeba by the formation of amoebastome (characteristic of amoeba phagocyte). This study showed that Acanthamoeba phagocytosis may be both an efficient means of obtaining nutrients for the amoeba and a significant factor in the pathogenesis of Acanthamoeba infections. Received: 2 April 2001 / Accepted: 12 April 2001     

Isolation and Genotyping of Acanthamoeba Strains from Soil Sources from Jamaica,West Indies

Acanthamoeba spp. are opportunistic pathogens that are ubiquitous in nature. Many species of this genus are responsible for a fatal encephalitis and keratitis in humans and other animals. Seventy‐two soil samples were collected from the parishes across Jamaica and assessed for the presence of Acanthamoeba spp. Cultivation was carried out on non‐nutrient agar plates seeded with heat killed Escherichia coli. PCR and sequencing of the DF3 region were carried out in order to genotype the isolated strains of Acanthamoeba. Thermotolerance and osmotolerance assays were utilized to investigate the pathogenic potential of the Acanthamoeba isolates. Acanthamoeba spp. was isolated from 63.9% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T4, T5, and T11. T4 genotype was most frequently isolated. Most isolates were thermotolerant or both thermotolerant and osmotolerant, indicating that they may present the potential to cause disease in humans and other animals.     

Distribution and possible interrelationships of pathogenic and nonpathogenicAcanthamoeba from aquatic environments

Among the more recently discovered agents of human disease are small, free-living amebae belonging to the generaNaegleria andAcanthamoeba. An overview of the distribution ofAcanthamoeba in recent surveys of the near shore waters of the northeastern United States is presented. There appears to be a particular association between the presence ofAcanthamoeba in marine sediments and the sites of oceanic sludge dumping. Amebae belonging to the genusNaegleria have not been isolated from these marine sediments which routinely yieldedAcanthamoeba. Starch gel electrophoretic analysis of enzymes suggests that some isolates ofAcanthamoeba from oceanic sludge dump-sites are not members of previously recognized pathogenic species.     

Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City

A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis.     

东海低氧区及邻近水域浮游植物的季节变化

根据2011年5月、8月、11月在东海低氧区及邻近水域(25°00'—33°30'N,120°00'—127°30'E)进行的多学科综合调查,对东海低氧区及邻近水域浮游植物群落结构特征及季节变化进行了相关研究。经Utermhl方法初步分析共鉴定出浮游植物4门74属248种(含变种、变型,不含未定种),主要由硅藻和甲藻组成,此外还有少量的金藻和蓝藻。春季优势种主要为具齿原甲藻(Prorocentrum dentatum)、柔弱伪菱形藻(Pseudo-nitzschia delicatissim)、骨条藻(Skeletonema sp.)和具槽帕拉藻(Paralia sulcata);夏季主要是中肋骨条藻(Skeletonema costatum)和海链藻(Thalassiosira sp.);秋季主要是具槽帕拉藻、圆筛藻(Coscinodiscus sp.)和柔弱伪菱形藻。调查区浮游植物平均细胞丰度在夏季最高,达到85.002×103个/L,春季次之,秋季最低。在水平方向上,春、夏两季,表层浮游植物细胞丰度在近岸出现高值,由近岸到外海细胞丰度逐渐降低;而在秋季则相反,在调查海域的东北部出现高值,随离岸距离的增加细胞丰度逐渐增加。在垂直方向上,春、夏两季,浮游植物细胞丰度在表层出现最大值,随着深度的增加细胞丰度逐渐降低;而在秋季细胞丰度分布比较均匀,随水深变化不明显。调查区表层浮游植物ShannonWiener多样性指数和Pielou均匀度指数的平面分布基本一致,并且与细胞丰度的分布大致呈镶嵌分布。调查浮游植物群落的演替规律是:从春季的甲藻(具齿原甲藻、微小原甲藻(Prorocentrum minimum)等)为主,硅藻(柔弱伪菱形藻、骨条藻等)为辅;演替至夏季的硅藻(中肋骨条藻、海链藻等)为主,甲藻(主要是梭状角藻(Ceratium fusus)和叉状角藻(Ceratium furca))为辅,到秋季进一步演替为硅藻(具槽帕拉藻、圆筛藻、柔弱伪菱形藻等)为主,铁氏束毛藻(Trichodesmium thiebaultii)为辅。浮游植物物种组成、优势种、细胞丰度及多样性指数均表现出明显的时空变化。低氧区与非低氧区浮游植物群集存在明显差异。     

Seasonal Distribution of Thermotolerant Free-Living Amoebae. I. Willard's Pond1

ABSTRACT. A quantitative study of the seasonal distribution of thermotolerant (37°C and 45°C), small free-living amoebae (FLA) was conducted in Willard's Pond, a warm, monomictic lake in the Piedmont region of South Carolina. Correlation of physical and chemical parameters with the seasonal distribution was facilitated by partitioning the aquatic ecosystem into benthic, planktonic, and neustonic habitats. Population densities of FLA peaked in late summer in each habitat; however, species composition varied between habitats. Littoral sediment appeared to be the major habitat for FLA, with peaks in populations of Acanthamoeba and Naegleria in August, Hartmannella in July, and Vahlkampfia in May. Populations in profundal sediment underwent dramatic seasonal shifts, apparently in response to the seasonal chemical changes in the hypolimnion. Acanthamoeba was most prevalent in late summer, representing as much as 82% of the FLA in profundal sediment. Distribution patterns and species composition of FLA from surface water were similar to those from littoral sediment; however, a greater percentage of Naegleria was found in surface water. Numerous FLA were isolated from the neustonic community (surface film), and the number of FLA isolated in the surface film at the deep water station was found to be significantly (P > 0.05) greater than the number from subsurface (5–10 cm) samples. In the water column, FLA populations consistently were highest in the detrital layer, which persisted at a depth of 3.0–3.4 m throughout the summer period. The large percentage of Naegleria contributing to FLA in the detrital layer suggests that Naegleria amoeboflagellates sink through the layer, flagellate, and swim back up, such migrations possibly being triggered by a reduction of nutrients below the layer or by the presence of anoxic, reducing conditions in the hypolimnion. In addition, weather events were found to play a major role in the redistribution of FLA between various habitats in the aquatic ecosystem, with such changes probably due to resuspension of FLA from littoral sediment by wind action and input from the watershed via runoff.     

Biological characterization of a clinical and an environmental isolate of <Emphasis Type="Italic">Acanthamoeba polyphaga</Emphasis>: analysis of relevant parameters to decode pathogenicity

Acanthamoeba spp. consists of free-living amoebae, widespread in nature, which occasionally can cause human infections including granulomatous amoebic encephalitis and amoebic keratitis. Acanthamoeba pathogenesis is not entirely known and correlations between pathogenic potential and taxonomy are complex issues. In order to decipher the definition of a pathogenic amoeba, the objective of this work was to decipher the definition of pathogenic amoeba by characterizing two isolates of Acanthamoeba polyphaga obtained from different origins (a keratitis patient and freshwater), looking for differences among them. The clinical isolate grew faster in Peptone-yeast extract-glucose (PYG) medium, transformed more rapidly from a trophozoite to cyst and exhibited increased cytopathic effect on cultured cells. Morphological differences were also noted, since freshwater amoebae presented more acanthopodia than the clinical isolate. Moreover, actin labeling demonstrated that microfilament organization varies between isolates, with the presence of locomotory structures as lobopodia and lamellipodia in the keratitis isolate, which were less adherent on plastic. Zymography demonstrated that the keratitis isolates presented higher proteolytic activity and also were more able to invade collagen matrices. Altogether, we conclude that a group of stable physiological characteristics exist in Acanthamoeba that can be related to pathogenicity.     

Diversity of Bacterial Endosymbionts of Environmental Acanthamoeba Isolates

Free-living amoebae are frequent hosts for bacterial endosymbionts. In this study, the symbionts of eight novel environmental Acanthamoeba strains isolated from different locations worldwide were characterized. Phylogenetic analysis revealed that they were related to one of four evolutionary lineages of amoeba symbionts recognized previously. This study provides evidence for the existence of only a small number of phylogenetically well-separated groups of obligate intracellular endosymbionts of acanthamoebae with global distribution.     

Breeding biology of Tenagomysis tasmaniae Fenton,Anisomysis mixta australis (Zimmer) and Paramesopodopsis rufa Fenton from south-eastern Tasmania (Crustacea: Mysidacea)

The number, size and developmental stage of young in the brood pouch of female Tenagomysis tasmaniae, Anisomysis mixta australis and Paramesopodopsis rufa was recorded throughout the year. Breeding was intensive from spring till the end of autumn for the three species. Calculation of the egg ratio for each species showed that their major reproductive peaks occurred during spring and summer. A winter depression in the breeding cycle was observed for T. tasmaniae and P. rufa, but A. mixta australis ceased breeding during winter. Seasonal variation in the length of gravid females and number of young carried was evident for these three species. Females were longer in spring and summer and carried more young than in autumn and winter. A linear relationship between female length and brood size was demonstrated for each species; annual and seasonal equations were calculated for females carrying each developmental stage. The seasonal equations showed that for a female of given length fecundity was greater during spring than any other season. Natality was estimated to be highest during late spring, summer and early autumn for the three species. No seasonal variation in the size of eggs was evident for the three species. The reproduction pattern of T. tasmaniae, A. mixta australis and P. rufa appears to be very similar to that reported for the majority of iteroparous coastal temperate mysids throughout the world.     

Seasonal Distribution of Thermotolerant Free-Living Amoebae. II. Lake Issaqueena1

A quantitative study of the seasonal distribution of thermotolerant (37°C and 45°C), small free-living amoebae (FLA) was conducted in Lake Issaqueena, a warm, monomictic lake with steep, sloping banks and a maximum basin depth of 10 m in the Piedmont region of South Carolina. Naegleria and Vahlkampfia were the most frequently encountered FLA in littoral sediment and surface water samples whereas Acanthamoeba was most commonly isolated from profundal sediment, especially during late summer. In the water column, FLA populations were highest in a persistent detrital layer; however, few amoebae were isolated from a massive (～1.5 m thick) layer of Oscillatoria. The only N. fowleri isolated in this study was from the detrital layer. Discussion of the influence of differences in watershed and basin morphology on variations in the size and generic composition of FLA populations for the aquatic ecosystems of Lake Issaqueena and Willard's Pond is included.     

Toxicity and Occurrence of Fusarium species and Mycotoxins in Late Harvested and Overwintered Grain from Norway, 1993

Samples of grain, harvested in October/November of 1993 or in the spring of 1994 from fields in Norway with overwintered grain, were collected. The presence of Fusarium species and the amount of mycotoxins produced by this genus were determined. The cytotoxic properties of the grain samples were examined with an in vitro methylthiazoltetrazolium (MTT)-cell culture assay using swine kidney and VERO cells as target cells. The total count of colony forming units of Fusarium species was about the same in the grain harvested in late autumn and in the spring, but the dominant species seemed to vary somewhat between the two groups. F. culmorum was found more in the samples from October/November while F. avenaceum was isolated more in the grain harvested in the spring. Only small amounts of F. sporotrichioides were present in the grain harvested in the autumn, and none was found in the overwintered grain. The deoxynivalenol content was significantly higher in the grain harvested in autumn than in the overwintered grain. Although very small amounts of toxins were detected in the overwintered grain, it was more cytotoxic than the grain harvested in October/November. A significant correlation between the cytotoxicity and the amount of F. avenaceum in the samples was found.