The Effect of Different Environmental Conditions on the Viability of Naegleria fowleri Amoebae

Naegleria fowleri, a free‐living amoeba found in soil and freshwater environments, is the causative agent of Primary Amoebic Meningoencephalitis. Infection occurs when amoebae enter the nasal cavity, attach to the nasal mucosa and travel along olfactory neurons towards the olfactory bulb. Upon reaching the central nervous system, the amoebae replicate very rapidly and can cause death in 3–10 days. Little is known about the conditions in which the amoeba can survive in the environment. We have tested conditions beyond the known boundaries on the viability of amoebae by introducing them into moderate and extreme salinity, pH, and temperatures. Our data shows that although viability expectedly decreases towards each of these extreme conditions, their tolerance was much greater than anticipated, including viability in moderate salinity, a wide pH range, and temperatures higher than the previously reported 45 °C.     

Modulation of Biological Functions of Naegleria fowleri Amoebae by Growth Medium

Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri , LEE, and a highly pathogenic strain, LEEmpCl, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri , LEE and LEEmpCl, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.     

Comparative Recoveries of Naegleria fowleri Amoebae from Seeded River Water by Filtration and Centrifugation

Detection of pathogenic Naegleria fowleri in environmental water samples, which is necessary for the prevention of primary amoebic meningoencephalitis, generally requires concentrating the samples. Two concentration techniques, filtration and centrifugation, were used to study the recovery of N. fowleri, in vegetative or cystic form, that had been mixed with the two other thermotolerant Naegleria species, N. lovaniensis and N. australiensis. Counting of amoebae was performed by the most probable number method on 10 water replicates of 100 ml and 10 ml each. With both concentration methods, recovery was better for cysts than for trophozoites (53% ± 21% versus 5% ± 5% by filtration and 57% ± 25% versus 22% ± 5% by centrifugation). The recovery of Naegleria trophozoites by filtration was very low, and centrifugation was significantly better than filtration in recovery of Naegleria trophozoites (22% ± 5% versus 5% ± 5%; P < 0.001). For cysts, however, filtration appeared as efficient as centrifugation, with equivalent values for recovery (53% ± 21% versus 57% ± 25%; P > 0.7). Although the recovery of cysts of N. fowleri obtained by filtration (51% ± 24%) appeared higher than that by centrifugation (36% ± 23%), the difference was not significant (P > 0.1). Both concentration methods have highly variable recovery rates, making accurate quantification of low concentrations (<100/liter) of N. fowleri in the environment difficult.     

Modulation of a "CD59-like" protein in Naegleria fowleri amebae by bacteria

Found in soil and freshwater habitats, Naegleria fowleri are free-living amebae that cause a fatal disease in humans called Primary Amebic Meningoencephalitis. In the natural environment, amebae feed on bacteria. In the infected host, the amebae lyse and ingest nerve tissue. Recently, we have established that N. fowleri expresses a "CD59-like" surface protein, but the function of this protein in the ameba has not been elucidated. In mammalian cells, CD59 is a complement-regulatory protein that inhibits complement-mediated lysis of cells expressing this protein. In the present study, expression of the "CD59-like" protein in response to bacteria and bacterial toxins was investigated by Western immunoblot analysis. Co-culture of N. fowleri with log phase Escherichia coli or Pseudomonas aeruginosa resulted in differential expression of the "CD59-like" protein. Co-cultures of amebae and bacteria were examined by electron microscopy. The results of our study implicate a possible protective role of the "CD59-like" protein in response to bacterial predators and bacterial toxins, because amebae remained intact after co-culture with bacteria.     

Identification of Naegleria fowleri and other Naegleria spp. (free-living amoebae) using cellulose acetate membrane electrophoresis of glucose phosphate isomerase

Abstract A simple isoenzyme cellulose acetate membrane electrophoresis method with respect to glucose phosphate isomerase (GPI) was developed for the differentiation of the human pathogenic free-living amoeba Naegleria fowleri from other Naegleria spp. A single GPI band was detected in all the species tested, the relative mobility of which could be used to identify N. fowleri . Of the other Naegleria spp., only N. italica and N. jadini shared a common GPI mobility. No intraspecies variation in GPI profile was detected, regardless of whether the strains were cultured in monoxenic or axenic media. The technique is proposed as a useful means of identifying N. fowleri soon after isolation from the environment.     

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.     

Rapid Detection and Enumeration of Naegleria fowleri in Surface Waters by Solid-Phase Cytometry

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.     

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.     

Detection of Naegleria fowleri cysts in environmental samples by using a DNA probe

Abstract In this study we tried to detect DNA Naegleria fowleri in artificially contaminated environmental samples, with or without sediments, containing 10⁴ cysts of this pathogenic amoeba. We used two assays to extract DNA from samples: first, direct DNA extraction, which gave positive results only for water samples without sediment; second, DNA extraction after sample incubation on agar plates, which allowed us to remove amoeba growing out of the sediments, and which gave positive results for all samples, even those initially with sediments (5, 500 or 500 mg). Thus, this molecular identification appears as a powerful tool to investigate N. fowleri growth in environmental samples.     

Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Abstract In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri , since Naegleriae ( N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini ) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, algae, y, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri .     

Light and Electron Microsopic Observations on the Pathogenesis of Naegleria fowleri in Mouse Brain and Tissue Culture

Two strains of pathogenic Naegleria were employed to infect mice and monkey kidney (Vero line) cell cultures. Mice were infected intranasally. Moribund mice were sacrificed and their brains processed for light and electron microscopy. The normal architecture of the infected brain was completely destroyed; the olfactory lobes and the cerebral cortex showed the heaviest damage. The inflammatory response was mainly in the form of neutrophil polymorphs (PMN) and was confined to the olfactory lobes and the superficial regions of cerebral cortex. Numerous amebas were seen interspersed with the degenerating neurons, glial processes, and PMN. Most conspicuous were the food vacuoles which contained host tissue in various stages of digestion. Amebas in the brain tissue also produced many micropinocytotic vesicles from the surface of the plasma membrane. These vesicles are interpreted as vehicles of transport of nutritive materials from the host tissue. The infected cell culture showed the characteristic cytopathic effect (CPE). The CPE was chiefly in the form of cell shrinkage, nuclear pycnosis and discontinuity of cell sheet. Amebas were often seen in an intracellular location. The Vero cells produced many fuzzy pinocytotic vesicles at these loci where the ameba plasma membrane and Vero cell membrane were in close apposition; the probable significance of this is discussed. Most impressive, however, were the pseudopodial formation and capturing of the host material which indicated the great phagocytic activity of the amebas. This was confirmed further by the presence of large numbers of food vacuoles containing host material in various stages of digestion. These observations show that the amebas invade and destroy the brain tissue by active phagocytosis.     

花生壳培养基对杏鲍菇菌丝生长的影响

以农业废料花生壳为主要原料设计不同配方,筛选适宜杏鲍菇菌丝生长的培养基及培养料.结果表明,母种培养基以m(花生壳):m(马铃薯)=3:1的配方生长最快,平均生长速度为4.53 mm/d,长势最旺盛;原种及栽培种培养料按m(棉籽壳):m(花生壳)=1:1的配方菌丝生长最快而健壮,生长速度可达5.43 mm/d.因此,在花...     

不同基本培养基及有机添加物对3个朵丽蝶兰品种组培苗生长的影响

研究不同基本培养基及有机添加物对3个品种朵丽蝶兰组培苗生长的影响。实验表明,朵丽蝶兰红花品种(Doritaenopsis Jiuhbao Red Rose)在以花宝1号作基本培养基的处理中,根粗、根长和叶长、双叶幅明显优于1/2 MS培养基;花宝1号培养基也利于黄花品种(Dtps.Chain Xen Queen)组培苗的叶片生长;添加香蕉泥100 g/L有利于3个品种即红花、黄花品种及白花红心品种Dtps.[I-Hsin Actor×(Tom Boy×Mount Beauty)]的根系生长,而添加香蕉泥70 g/L+马铃薯30 g/L仅有利于黄花品种的叶片生长。     

Molecular cloning of, and phylogenetic analysis of, an actin in Naegleria fowleri

Abstract Two strains of Lactobacillus acidophilus Group A1, the neotype ATCC 4356 and a human isolate NCFM-N2, widely used as a dietary adjunct in milk and cultured dairy products, were transformed with plasmid DNA by electroporation. The transformation characteristics exhibited by the two L. acidophilus strains were found to differ markedly even though they appeared similar at the genomic level based on the DNA patterns of Sma I restriction fragments. To our knowledge, this is the first report of a consistent, reproducible transformation system of Lactobacillus acidophilus strains comprising the A1 DNA homology group.     

Computational identification of putative miRNAs and their target genes in pathogenic amoeba Naegleria fowleri

Naegleria fowleri is a parasitic unicellular free living eukaryotic amoeba. The parasite spreads through contaminated water and causes primary amoebic meningoencephalitis (PAM). Therefore, it is of interest to understand its molecular pathogenesis. Hence, we analyzed the parasite genome for miRNAs (microRNAs) that are non-coding, single stranded RNA molecules. We identified 245 miRNAs using computational methods in N. fowleri, of which five miRNAs are conserved. The predicted miRNA targets were analyzed by using miRanda (software) and further studied the functions by subsequently annotating using AmiGo (a gene ontology web tool).     

Single-cell isolation and cloning of Tetrahymena thermophila cells with a fluorescence-activated cell sorter

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.     

极地雪藻在不同培养基中生长和虾青素累积的研究

在常温（23℃）和低温（10℃）条件下,用BBM、Bold 1NV、TAP和MCM四种培养基对极地雪藻进行培养。通过对生长速率、细胞数、A535值及虾青素累积量的测定,比较不同培养基、不同培养条件对极地雪藻的生长与虾青素累积的影响。结果表明,低温有利于极地雪藻的生长和虾青素的累积,BBM培养基比其它培养基更适合极地雪藻的生长,10℃时条件下其生长速率最高,培养14d细胞数可达到3．53&#215;10^6／mL;在高光强条件下培养15d后用BBM培养基培养的极地雪藻细胞的虾青素累积为其它培养基的2．21-3．59倍。     

Analysis of the behaviour of<Emphasis Type="Italic"> Dictyostelium discoideum</Emphasis> in immobilised state by means of continuous cultivation

The social amoeba Dictyostelium discoideum is amenable to cultivation in the immobilised state most simply by colonisation of porous supports. An analysis of the growth behaviour of D. discoideum in the immobilised state is reported. For this purpose, D. discoideum was cultivated in continuously operated reactors in a suspension culture (homogeneous system) and immobilised on a porous support (heterogeneous system). Thus, it is possible to compare homogeneous and heterogeneous systems under steady-state conditions. Immobilisation was achieved by the colonisation of porous glass beads (SIRAN). Simple models are applied in order to describe the growth behaviour of fractions of both the cells in free suspension and the cells inside the porous carrier. This analysis shows that D. discoideum inside the pores grows at a rate of only about 10% compared with that in free solution. The consequence of this behaviour is discussed in terms of reactor performance.     

培养基及其组成对水母雪莲悬浮培养细胞生长及黄酮形成的影响

8种不同的基本培养基对水母雪莲(Saussurea medusa)细胞生长和黄酮形成的影响不同。实验结果表明,MS培养基较有利于细胞生长和黄酮形成。从MS培养基修饰得到的MG和MP培养基比前者细胞生长量与黄酮产量分别提高32%和70%。碳源、氮源、植物激素对细胞生长及黄酮形成的影响较为明显。用HPLC对细胞培养物中两种有效黄酮(Jaceosidin和Hispidulin)作了定性定量分析。     

Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media

Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps. This revised version was published online in June 2006 with corrections to the Cover Date.