Cytopathogenicity of Naegleria fowleri for cultured rat neuroblastoma cells

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to 51Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44 degrees C for 60 min or at 60 degrees C for 30 min. Cytotoxicity was stable during storage at 4 degrees C or at -20 degrees C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4 degrees C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

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.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

Activated macrophages demonstrate direct cytotoxicity, antibody-dependent cellular cytotoxicity, and enhanced binding of Naegleria fowleri amoebae

Macrophages activated in vivo by injection of Corynebacterium parvum or bacillus Calmette-Guérin caused direct cytolysis of the pathogenic free-living amoeba, Naegleria fowleri, in vitro. Amoebicidal activity was time and cell density-dependent but was not dependent on the presence of specific antibody. Antibody-dependent cellular cytotoxicity for amoebae was also expressed by activated macrophages. Resident and thioglycolate-elicited macrophages demonstrated low cytolytic activity under all conditions tested. From scanning electron microscopy it appears that the degree of target cell binding is directly related to the degree of cytolysis expressed by the macrophage populations. Cell-cell contact was required for cytolysis of amoebae by activated macrophages since cytolysis did not occur when contact was blocked by a porous filter. For each macrophage population, the levels of amoebicidal activity and tumoricidal activity were comparable.     

Cytopathogenicity of Naegleria fowled for Cultured Rat Neuroblastoma Cells1

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to ⁵¹Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44°C for 60 min or at 60°C for 30 min. Cytotoxicity was stable during storage at 4°C or at ?20°C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4°C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

Cocultivation of the amoeba Naegleria fowleri and the amoebicin- producing strain Bacillus licheniformis M-4.

Antagonism between Bacillus licheniformis M-4 and the pathogenic amoeba Naegleria fowleri HB-1 during cocultivation was influenced by the composition of the medium and the initial amoeba/bacterium ratio. While a ratio of 50 caused complete lysis of amoebae in soil extract with 0.3% glucose (SEG) before 72 h, this ratio had to be at least 12-fold lower in order to obtain similar results in Cline medium. Sporulation of B. licheniformis M-4 took place much earlier in SEG. Amoebicin production was stimulated by the presence of amoebae by either shortening the time of production (as in SEG) or increasing the amount of amoebicins released (as in Cline medium). Electron microscopy showed that amoebae cocultivated in the Cline medium contained bacteria enclosed in digestive vacuoles, while amoebae from SEG cocultures did not.     

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

In vitro migration of human large granular lymphocytes

Large granular lymphocyte (LGL)-enriched peripheral blood mononuclear cells were prepared on discontinuous gradients of Percoll. Migration into nitrocellulose filters was studied in a 2-hr assay with the use of modified Boyden chambers. LGL migrated into filters in response to casein or C5a. Migration depended on the presence of a concentration gradient of chemoattractant between the lower and upper compartment of the chambers. Percoll-purified high density small lymphocytes had little or no migratory capacity under these conditions, requiring a longer incubation time (4 hr) for consistent migration. Migratory capacity in response to stimuli correlated with the frequency of LGL in the various fractions of Percoll gradients. Fractionation of LGL-enriched Percoll preparations with monoclonal antibodies and immunoadsorbent columns or a cell sorter revealed that cells with migratory capacity in response to stimuli were B73.1+ and OKT3-, thus confirming that migrating cells were LGL. LGL preincubated with interferon (IFN) showed enhanced spontaneous motility but no increased responsiveness to chemoattractants. IFN did not modify the migratory capacity of small lymphocytes. The prompt migration of LGL in response to stimuli is consistent with the hypothesis that these cells may serve as one of the first easily mobilizable lines of resistance against infectious agents and tumors. The migration assay described here may offer a better insight into the functions and regulation of LGL.     

Cytolytic activity of Naegleria fowleri cell-free extract

The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30-60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by 51Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50 degrees C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing 51Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Biological factors affecting enflagellation of Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate that can be evoked to transform from amoebae to flagellates by subculture to nonnutrient buffer. More than half of the amoebae of strains KUL, nN68, and Lovell became enflagellated 300 min after subculture to amoeba-saline, whereas no amoebae of strains NF66, NF69, and HB4 did. N. fowleri nN68 enflagellated best when grown at 32 or 37 degrees C and subcultured to amoeba-saline at 37 or 42 degrees C. Amoebae from the stationary phase of growth enflagellated more readily than did actively growing amoebae. Incubation in expended culture medium from stationary-phase cultures enhanced the capability of growing amoebae to enflagellate after subculture to amoebasaline. Enflagellation was more extensive when the population density in amoebasaline did not exceed 2 x 10(5) amoebae per ml. Cycloheximide at 1 microgram/ml and actinomycin D at 25 micrograms/ml inhibited growth of N. fowleri nN68. Cycloheximide at 0.5 microgram/ml and actinomycin D at 25 micrograms/ml completely prevented enflagellation when added at time zero. Cycloheximide at 0.5 microgram/ml, added 120 to 300 min after initiation of enflagellation, prevented further differentiation and caused existing flagellates to revert to amoeboid cells. Similarly, actinomycin D at 25 micrograms/ml, added 90 to 300 min after initiation of enflagellation, retarded differentiation and caused flagellates to revert. Radiolabeled precursors were incorporated into macromolecules during differentiation in nonnutrient buffer. Enflagellation of N. fowleri is a suitable model for studying regulation of a eucaryotic protist.     

Occurrence and pathogenicity of Naegleria fowleri in artificially heated waters

The occurrence of pathogenic Naegleria fowleri in thermal discharges, recipient waters, and cooling towers of eight power plants located in western Pennsylvania was investigated for 2 years in conjunction with several environmental measurements. Pathogenic N. fowleri was detected in one cooling tower and in the discharge, receiving waters, or both of five of eight localities. The occurrence of this organism was related to elevated temperatures, but no significant correlation was found for other biological and chemical parameters. Laboratory experiments on the effect of pH on pathogenic N. fowleri documented 100% survival at a range from 2.1 to 8.15. Higher pH reduced or killed the amoebae. No case of human primary amoebic meningoencephalitis occurred during the study.     

Occurrence and pathogenicity of Naegleria fowleri in artificially heated waters.

The occurrence of pathogenic Naegleria fowleri in thermal discharges, recipient waters, and cooling towers of eight power plants located in western Pennsylvania was investigated for 2 years in conjunction with several environmental measurements. Pathogenic N. fowleri was detected in one cooling tower and in the discharge, receiving waters, or both of five of eight localities. The occurrence of this organism was related to elevated temperatures, but no significant correlation was found for other biological and chemical parameters. Laboratory experiments on the effect of pH on pathogenic N. fowleri documented 100% survival at a range from 2.1 to 8.15. Higher pH reduced or killed the amoebae. No case of human primary amoebic meningoencephalitis occurred during the study.     

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

A paper membrane filter assay for ciliate chemoattraction

A quantitative bioassay for ciliate chemoattraction based on the Boyden assay is described with the ciliates Tetrahymena thermophila and Tetrahymena pyriformis as test organisms. A chamber is separated into two compartments by a Whatman 3MM filter, and a suspension of starved cells (approximately 10(5) cells/ml) is placed in one compartment and a solution containing attractant in the other. The gradient of chemoattractant across the filter causes the cells to swim through the filter into the attractant-containing compartment where their appearance is determined by electronic cell counting. The assay described is convenient with a signal-to-noise ratio of approximately 10. It is shown here to work with the attractants proteose peptone and platelet-derived growth factor.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

A biochemical comparison of proteases from pathogenic naegleria fowleri and non-pathogenic Naegleria gruberi

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis (PAM). Proteases have been suggested to be involved in tissue invasion and destruction during infection. We analyzed and compared the complete protease profiles of total crude extract and conditioned medium of both pathogenic N. fowleri and non-pathogenic Naegleria gruberi trophozoites. Using SDS-PAGE, we found differences in the number and molecular weight of proteolytic bands between the two strains. The proteases showed optimal activity at pH 7.0 and 35 degrees C for both strains. Inhibition assays showed that the main proteolytic activity in both strains is due to cysteine proteases although serine proteases were also detected. Both N. fowleri and N. gruberi have a variety of different protease activities at different pH levels and temperatures. These proteases may allow the amoebae to acquire nutrients from different sources, including those from the host. Although, the role of the amoebic proteases in the pathogenesis of PAM is not clearly defined, it seems that proteases and other molecules of the parasite as well as those from the host, could be participating in the damage to the human central nervous system.     

The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis

From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.     

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14⁺ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.