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At least 19 recordsLinked to original sources

PubMed1988

An in vitro comparative study on the effect of amphotericin B, econazole, and 5-fluorocytosine on Naegleria fowleri, Naegleria australiensis, and Naegleria australiensis s.sp. italica.

We tested in vitro amphotericin B (AMP-B), econazole (ECO), and 5-fluorocytosine (5-FC) on pathogenic Naegleria fowleri (KUL strain), Naegleria australiensis s.sp. italica (AB-T-F3, original strain) to assess their sensitivity to chemotherapeutic compounds. Previous reports have shown the polyene antibiotic AMP-B to be the most active agent. It was, however, much more active on N. fowleri than on N. australiensis and N. australiensis s.sp italica. 5-FC and ECO gave rise to non appreciable effect at non-toxic corresponding dosages in vivo. The results of these in vitro tests are discussed.

Amphotericin B↗

PubMedDate not supplied

Isolation of a unique membrane protein from Naegleria fowleri.

Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.

Amebiasis↗

PubMed2025

Eating the brain - A multidisciplinary study provides new insights into the mechanisms underlying the cytopathogenicity of Naegleria fowleri.

Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis (PAM), requires increased research attention due to its high lethality and the potential for increased incidence as a result of global warming. The aim of this study was to investigate the interactions between N. fowleri and host cells in order to elucidate the mechanisms underlying the pathogenicity of this amoeba. A co-culture system comprising human fibrosarcoma cells was established to study both contact-dependent and contact-independent cytopathogenicity. Proteomic analyses of the amoebas exposed to human cell cultures or passaged through mouse brain were used to identify novel virulence factors. Our results indicate that actin dynamics, regulated by Arp2/3 and Src kinase, play a considerable role in ingestion of host cells by amoebae. We have identified three promising candidate virulence factors, namely lysozyme, cystatin and hemerythrin, which may be critical in facilitating N. fowleri evasion of host defenses, migration to the brain and induction of a lethal infection. Long-term co-culture secretome analysis revealed an increase in protease secretion, which enhances N. fowleri cytopathogenicity. Raman microspectroscopy revealed significant metabolic differences between axenic and brain-isolated amoebae, particularly in lipid storage and utilization. Taken together, our findings provide important new insights into the pathogenic mechanisms of N. fowleri and highlight potential targets for therapeutic intervention against PAM.

Naegleria fowleri↗

PubMed1993

[Interleukin-2 production and alteration of T cell subsets in mice infected with Naegleria fowleri].

Naegleria fowleri is the cause of primary amoebic meningoencephalitis in man. IL-2 levels after stimulation of T lymphocytes by PHA or N. fowleri lysates, the amounts of T lymphocyte subsets and the blastogenic responses of T lymphocytes in mice after infected with pathogenic N. fowleri were studied comparing between two study groups, one 1 x 10(4) trophozoites inoculated mice and the other 1 x 10(5) trophozoites inoculated mice. All experimental samples were obtained on the day 7, 14 and 24 after inoculation. The mice inoculated with 1 x 10(4) trophozoites showed a 14.3% mortality rate, and 72.2% in the mice inoculated with 1 x 10(5) trophozoites. The IL-2 levels on day 14 of two experimental groups were significantly decreased as compared with the control group. Thy 1.2+ T cells in the total spleen lymphocytes of 1 x 10(5) trophozoites inoculated group on day 7 were significantly increased compared with the control group. There was no significant difference between 1 x 10(4) trophozoites inoculated group and the control group. L3T4+ T cells and Ly2+ T cells in the total spleen lymphocytes of 1 x 105 trophozoites inoculated group on day 7 were significantly increased compared with the control group. The DNA S fraction of T cells in the spleen of 1 x 10(5) trophozoites inoculated group was significantly increased on day 7. The amount of S fractions of DNA were sequentially decreased on day 14 and 24, but they were also significantly increased compared with the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Amebiasis↗

PubMed2002

Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a fatal disease of the central nervous system that is acquired while swimming or diving in freshwater. A cDNA clone designated Mp2C15 obtained from N. fowleri was used as a probe to distinguish N. fowleri from other free-living amoebae. The Mp2C15 probe hybridized to genomic DNA from pathogenic N. fowleri and antigenically related non-pathogenic N. lovaniensis. Mp2C15 was digested with the restriction enzyme XbaI, resulting in two fragments, Mp2C15.G and Mp2C15.P. Four species of Naegleria and four species of Acanthamoeba were examined for reactivity with Mp2C15.P. Mp2C15.P was specific for N. fowleri and was used in the development of a nested PCR assay which is capable of detecting as little as 5 pg of N. fowleri DNA or five intact N. fowleri amoebae. In summary, a rapid, sensitive, and specific assay for the detection of N. fowleri was developed.

Amebiasis↗

PubMed1983

Comparison of Naegleria fowleri and Naegleria gruberi cultivated in the same nutrient medium.

The human pathogenic amoeboflagellate Naegleria fowleri and the nonpathogenic species N. gruberi can be cultivated axenically but usually in different media. Naegleria fowleri 6088 has been adapted to grow in Balamuth H-4 medium, usually used to propagate N. gruberi nB81, and nB81 has been adapted to grow in supplemented Nelson's medium, usually used to propagate N. fowleri. N. gruberi nB81, grown in either medium, enflagellated 135 to 150 min after subculture to non-nutrient amoeba saline, whereas 6088 required 225 min. Naegleria gruberi nB81 grown in either medium was agglutinated by 100 micrograms concanavalin A/ml, whereas N. fowleri 6088 was not. Naegleria fowleri and N. gruberi grown in Nelson's medium became rounded to a greater extent upon chilling at 5 degrees C and remained rounded longer than Naegleria grown in Balamuth medium. The specificity of the surface antigens was an inherent characteristic of each species and not dependent upon the propagating medium, but Naegleria grown in Nelson's medium was agglutinated more reproducibly and more effectively by antiserum. N. gruberi was somewhat more resistant to acriflavine, actinomycin D, cycloheximide, or tetracycline than N. fowleri, regardless of the culture medium. Naegleria fowleri 6088 grown in Nelson's medium, however, was more resistant to actinomycin D, daunomycin, mithramycin, sulfamethoxazole, or tyrocidine than 6088 grown in Balamuth medium. There are limitations on the validity of comparisons of N. fowleri and N. gruberi based upon cultures grown in different media.

Agglutination Tests↗

PubMed1990

Ultrastructural studies on the effect of tumor necrosis factor on the interaction of neutrophils and Naegleria fowleri.

Naegleria fowleri is the common etiologic agent of primary amebic meningoencephalitis (PAM). We investigated the interaction of human neutrophils with Naegleria trophozoites and examined the effect of neutrophil stimulation by the recombinant human tumor necrosis factor (TNF) on this interaction. As indicated by scanning and transmission electron microscopy, TNF stimulated the adherence of neutrophils to N. fowleri with destruction of the ameba. Neutrophil iodination, an indirect measure of stimulation, increased from 0.81 +/- 0.23 nmol/10(7) cells/hr to 2.41 +/- 0.62 nmol/10(7) cells/hr following the addition of TNF to the neutrophil-N. fowleri mixture (P less than 0.05). This was independent of complement or specific immunoglobulin. Ingestion of neutrophils by Naegleria trophozoites was observed following more prolonged incubation, particularly in the absence of TNF. These findings suggest a role for TNF-mediated destruction of Naegleria trophozoites by neutrophils in host defense, and that ingestion of host neutrophils by Naegleria trophozoites may represent a virulence factor.

Animals↗

PubMed1980

Activation of the alternative complement pathway by Naegleria fowleri.

Naegleria fowleri amoebae were lysed by adult fresh human serum, and their multiplication was inhibited in culture medium supplemented with 10% fresh human serum. Heat inactivation (56 degrees C, 30 min) of serum abrogated these lytic and inhibitory effects. Absorption of human serum with amoebae failed to reduce immunoglobulin levels, and no specific antibody was detected in untreated or treated sera by counterimmunoelectrophoresis. Conversion of C3 and C3i occurred after incubation of n. fowleri with serum which had been treated with ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid, indicating activation of complement via the alternative pathway.

Adolescent↗

PubMed1982

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.

Amoeba↗

PubMed1984

Human neutrophils require activation by mononuclear leucocyte conditioned medium to kill the pathogenic free-living amoeba, Naegleria fowleri.

Naegleria fowleri is a free-living amoeba which causes a fulminant and rapidly fatal meningoencephalitis in man. Human neutrophils fail to kill the amoeba in vitro, but can do so if they are exposed to conditioned medium (CM) from PHA stimulated mononuclear leucocytes (MNLs). Specific antibody or complement was required to effect amoeba killing by CM modified neutrophils. Only short time exposure of the leucocytes to CM was required to endow them with amoebicidal properties. The CM was also shown to contain neutrophil migration inhibition activity and an activity(ies) which induced a respiratory burst in neutrophils. The results highlight the importance of MNL products other than specific antibody in neutrophil anti-microbial activity.

Amoeba↗

PubMed1982

Intravenous infection of mice with Naegleria fowleri.

Naegleria fowleri produced fatal meningoencephalitis in mice following intravenous (i.v.) inoculation. Amebae were present in the peripheral circulation for 120 minutes after i.v. inoculation with a dose of 10(7) trophozoites per mouse. Amebae were cultured from and observed in brain (days 1-21), lung (days 1-12), and liver and kidney (days 1-5). Infected mice exhibited weight loss, leukocytosis, reduced lymphocyte/neutrophil ratio, neurologic symptoms, and mortality. Histologically, the disease was characterized by an acute, hemorrhagic, necrotizing meningoencephalitis. Although amebae were detected in tissues other than brain, pathologic involvement of these tissues was minimal.

Amebiasis↗

PubMed1982

Cytopathogenicity of Naegleria fowleri and Naegleria gruberi for established mammalian cell cultures.

Amebae of Naegleria fowleri and Naegleria gruberi were cytopathic for nine established mammalian cell cultures, including mouse and human fibroblasts, rabbit and monkey kidney cells, rat and mouse neuroblastoma cells, baby hamster kidney cells, and human epithelioma and carcinoma cells. Nine strains of N. fowleri were equally cytopathic for rodent neuroblastoma cells. As few as one ameba per million neuroblastoma cells destroyed the mammalian target cells after 9 days. The N. fowleri grew and destroyed rat neuroblastoma cells at 30 to 37 C whereas N. gruberi grew and destroyed the target cells at 25 to 30 C. Both N. fowleri and N. gruberi attached efficiently to the target cells at 30 to 37 C; N. gruberi but not N. fowleri attached efficiently at 25 C. Electron microscopic observations of mixed cultures of N. fowleri and neuroblastoma cells established that the amebae, after 12 hr, had ingested portions of the neuroblastoma target cells without causing cell lysis. Conversely, N. gruberi amebae, after attaching to target cells, disrupted the plasma membrane and cytoplasm of the target cells although the target cell nucleus remained intact. The amebae then ingested the target cell debris.

Amoeba↗

PubMed1987

Serology of Naegleria fowleri and Naegleria lovaniensis in a hospital survey.

An avidin-biotin horseradish peroxidase method was used to detect antibodies to Naegleria fowleri and N. lovaniensis in human serum samples. Antibodies were detected in 101 specimens from 115 hospital patients ranging in age from 15 to 98 years. Class-specific anti-immunoglobulins identified antibodies as IgG and IgM. IgG antibody titers to both species ranged from 1:20 to 1:640. Seven of 15 serum samples collected from newborn infants also demonstrated IgG antibodies to these organisms with a titer range of 1:20 to 1:80. The immunoperoxidase test and Western blot analysis of selected serum samples demonstrated a close similarity in serological results between N. fowleri and N. lovaniensis.

Adolescent↗

PubMed1983

Agglutination of Naegleria fowleri and Naegleria gruberi by antibodies in human serum.

The capability of serum samples from 423 human subjects to agglutinate rounded cells of Naegleria fowleri nN68 was assessed. Sera from the umbilical cords of seven infants failed to agglutinate N. fowleri cells. The median agglutination titer was 1:4 for sera from children through age 4 years, 1:8 for sera from juveniles 5 to 15 years of age, and 1:16 for sera from subjects 15 to 30 years old. The agglutination titers of sera from older adults decreased to a median of 1:8 for the 40- to 60-year-old age group and to 1:4 for the 60- to 90-year-old subjects. Serum samples from young adults agglutinated rounded cells of both N. fowleri and N. gruberi. The agglutination activity for N. fowleri was removed by absorption with N. fowleri but not with N. gruberi. Conversely, agglutination activity for N. gruberi was removed by absorption with N. gruberi but not with N. fowleri. The agglutinating activity for N. fowleri was immunoglobulin M. Serum samples from children displayed markedly disparate capabilities to agglutinate N. fowleri and N. gruberi. Only rounded cells of N. fowleri or N. gruberi were reliably agglutinated by human serum samples. Live or paraformaldehyde-killed cells could be used in the assay, but live N. gruberi cells returned to the amoeboid form, and these agglutinated poorly.

Adolescent↗

PubMed1993

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

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, Gram-negative bacteria, algae, 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.

Animals↗

PubMed1977

Immunization of mice against Naegleria fowleri infection.

Naegleria fowleri produces fatal meningoencephalitis in humans and in experimentally infected laboratory animals. The course of the disease in mice is dependent upon the infecting dose of amoebae, route of inoculation, and prior exposure to Naegleria antigens. DUB/ICR mice were immunized by various routes and antigen preparations, held for 21 days, and, together with noninfected control mice, challenged intravenously (i.v.) or intranasally (i.n.) with 10(7) or 10(6) N. fowleri per mouse, respectively. Mice immunized with liver or formalinized N. fowleri or live N. gruberi subcutaneously, intraperitoneally, i.v., or i.n. were significantly protected against a subsequent lethal challenge with N. fowleri i.v. or i.n. In general, i.v. inoculation afforded greated protection than other routes of immunization, intact cells immunized mice better than did cell fragments, and N. gruberi appeared to be a better immunogen than N. fowleri.

Administration, Intranasal↗

PubMed1976

Modification of resistance of mice to Naegleria fowleri infections.

Naegleria fowleri, which produces a fatal meningoencephalitis in humans, is also able to produce a progressive and fatal disease in mice. The course of the disease in DUB/ICR mice is dependent upon the infecting dose of organisms, whether administered intraperitoneally (i.p.) or intravenously (i.v.). All of the mice receiving 10(7) trophozoites/mouse i.v. or 4.85 X 10(7) trophozoites/mouse i.p. were killed within 10 days. Escherichia coli O26:B6 lipopolysaccharide, administered at a dose of 1 mg/kg 24 h prior to N. fowleri, afforded some protection for several days after challenge, but by day 8 there was no difference in survival of untreated and endotoxin-treated mice. No significant protection was afforded by a complex of lipid A with concanavalin A (ConA) or bovine serum albumin (BSA) or by dimethylmyristamide-BSA, dimethylmyristamide, BSA, beta-hydroxymyristic acid-ConA, beta-hydroxymyristic acid, ConA, myristic acid-BSA, or myristic acid. Mice surviving primary i.v. or i.p. challenge doses of N. fowleri, 5 X 10(6) and 10(7) trophozoites/mouse, respectively, were highly resistant to rechallenge with an i.v. dose of organisms (5 X 10(6) Naegleria/mouse) that produced uniformly fatal disease in untreated control mice.

Amoeba↗

PubMed1983

Host resistance of mice to Naegleria fowleri infections.

Naegleria fowleri is an etiological agent of primary amoebic meningoencephalitis in humans and laboratory animals. The determinative factors in host resistance of mice to N. fowleri infections have not been fully characterized. Male or female B6C3F1 mice stimulated by intraperitoneal administration of 10(6) amoebae of N. fowleri nN68 per mouse produced agglutinating activity and markedly elevated levels of serum and immunoglobulins M and G. Despite a marked humoral response, protective immunity was increased only marginally by active immunization. Host resistance was not impaired by prior treatment with 350 rads of 60Co radiation or 200 mg of cyclophosphamide per kg or by concurrent daily treatment with 30 mg of cyclophosphamide per kg for 14 days. Moreover, host resistance was not impaired by daily treatment with 4 mg of diethylstilbestrol per kg for 14 days, with challenge on day 2 of drug exposure or 24 h after the last drug treatment. Mice depleted of hemolytic complement by cobra venom factor were more susceptible to N. fowleri infection than were untreated mice.

Agglutination Tests↗