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PubMed2025

Detection and identification of Naegleria species along with Naegleria fowleri in the tap water samples.

Naegleria fowleri, the causative agent of Primary Amoebic Meningoencephalitis (PAM), is commonly found in warm freshwater environments and can enter the brain through nasal passages during activities like swimming or ablution. PAM has a high fatality rate, raising concerns about its global health impact. In Pakistan, particularly in Karachi, a significant number of cases have been reported, often with no history of recreational water exposure, but with regular ablution using tap water. This study analyzed the physicochemical parameters, abundance of total and fecal coliforms, and detected N. fowleri and other Naegleria species in tap water samples from Karachi using PCR with ITS- and Naegl-primers. Almost all samples exhibited high temperatures, low chlorine levels, and a high presence of coliforms. N. fowleri and other Naegleria species were detected in 11 out of 39 samples. Sequence analysis identified N. fowleri in tap water from the Golimar and Lyari areas of Karachi, while the other nine samples revealed different Naegleria species. This study suggests that the combination of high temperatures, insufficient chlorination, and the presence of coliforms may create favorable conditions for N. fowleri growth. However, these factors are not exclusive to the Golimar and Lyari areas, indicating that other environmental or infrastructural factors, not detailed in this study, may have contributed to the presence of N. fowleri in that specific location.

Naegleria fowleri↗
PubMed1979

Pathogenesis of pathogenic Naegleria amoeba.

In brain sections of the Naegleria-caused cases of primary amoebic meningoencephalitis, extensive demyelinization was found in the white matter, besides the severe histopathological changes and large clusters of trophozoites in the grey matter. The myelinoclasis appeared to be a result of a specific phospholipolytic effect, unlike that in post-viral encephalomyelitis, which has been attributed to vascular blockade or hemorrhages. In monkey kidney cell cultures a very early cytopathic effect was observed and traced to the cytolytic property of the seeding culture fluid. Rat brain slices inoculated with Naegleria culture exhibited amoebic growth and demyelinization in 28-52 hours incubation at 35 degrees C. In a chemically defined medium containing sphingomyelin, casein and glucose, the Naegleria produced a limited growth parallelling the clearance of the lipid turbidity during a 72 hour incubation at 35 degrees C. Chromatographic analysis of the turbidity-cleared cultures revealed decomposition of sphingomyeline with liberation of choline, sphingosine and fatty acids. It is, hence, concluded that the pathogenicity of cytopathic effect of pathogenic Naegleria can be attributed to the latter's capacity to liberate a phospholipolytic enzyme or factor during active growth, which "makes holes" in the lipid-rich cytoplasmic membrane of cells as well as demyelinizes nerve tissue.

Amebiasis↗
PubMed1979

Observations by immunofluorescence microscopy and electron microscopy on the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures.

The destruction of secondary mouse-embryo (ME) cells by Naegleria fowleri was studied by indirect immunofluorescence with ME-cell antiserum as a specific label to trace the fate of mammalian-cell cytoplasm. The appearance of naegleria-induced cytopathic effect in the cultures coincided with the accumulation of discrete particles containing granules of ME-cell antigen within the cytoplasm of amoebae, suggesting that the organisms ingested host-cell material. In cultures containing cytochalasin B, a non-lethal inhibitor of phagocytosis by N. fowleri trophozoites failed to acquire any granular fluorescence and were not cytopathogenic. The engulfment of mammalian-cell cytoplasm by the organisms was confirmed when thin sections of naegleria-infected ME-cell cultures were examined by electron microscopy. Amoebae were seen in the process of detaching portions of cytoplasm from whole ME cells by means of distinctive ingesting pseudopodia, and fragments of mammalian-cell cytoplasm were identified within the food vacuoles of trophozoites. There was no evidence for cytotoxic disruption of ME cells before or during engulfment of these fragments. It is concluded that N. fowleri trophozoites attack and destroy cultured ME cells by a phagocytosis-like mechanism alone, without the aid of any amoeba-associated cytotoxic or cytolytic agents. The possible significance of these findings with respect to the in-vivo pathocity of N. fowleri is discussed.

Amoeba↗
PubMed1979

Starch gel electrophoresis: an effective method for separation of pathogenic and nonpathogenic Naegleria strains.

Isoenzyme electrophoresis of 7 different enzyme systems was used to compare 24 strains of Naegleria fowleri and 6 strains of N. gruberi. The 30 strains could be grouped into 4 distinct categories based upon zymogram patterns. No interstrain band variation in all enzyme systems was demonstrated in pathogenic strains of N. fowleri. Three nonpathogenic high temperature-tolerant strains of Naegleria had similar zymograms. Four of the 5 remaining nonpathogenic Naegleria strains had no interstrain band variation. Based upon zymograms, the 22 pathogenic strains constitute a homogenous species. Similarly the high temperature-tolerant nonpathogenic strains formed a cohesive group. The remaining nonpathogenic strains could be separated into 2 groups.

Acid Phosphatase↗
PubMed1975

The effects of some factors on the growth and morphology of Naegleria sp. and three strains of the genus Acanthamoeba.

The effects of various biophysical and chemical factors on the cytology of vegetative stages of Naegleria sp., Vitek strain, Acanthamoeba culbertsoni, Acanthamoeba castellanii, Neff strain and Acanthamoeba polyphaga, No. 1289, were studied. The amoebae were cultured in a liquid medium under axenic conditions. The optimum temperature was 37 degrees C for pathogenic strains of Naegleria sp. and Acanthamoeba culbertsoni and 20 degrees C for A. castellanii. No changes were observed in the growth of A. polyphaga at the temperatures 20 degrees and 37 degrees C. The strains investigated grew at pH values of 5.6 to 7.7 using Soerensen's buffer. At the limit values the growth was inhibited and the morphology of cells was markedly changed. All of the four strains grew still at pH 8.4 kept by NaHCO3. A. polyphaga grew at partial anaerobiosis. The three tested strains of the genus Acanthamoeba grew in liquid axenic medium with 0.89% NaCl. The growth of Naegleria sp., Vitek was inhibited already at 0.2% concentration of this salt. The addition of 3 X 10(-2) m KCl to the culture medium had a harmful effect on the growth and morphology of three tested strains, except A. polyphaga. In the culture medium containing 2 X 10(-3) m CaCl2 the encystment of both pathogenic strains was stimulated. The cytological changes under experimental conditions were manifested by atypical movement of trophozoits and their intracellular structure.

Amoeba↗
PubMed1979

Inhibition by amoeba-specific antiserum and by cytochalasin B of the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures.

Inhibitors of trophozoite motility and phagocytosis were used to investigate the mechanism of Naegleria fowleri cytopathogenicity in mouse-embryo (ME)-cell cultures. Amoebae that were immobilised and agglutinated by specific antiserum exhibited no cytopathic activity, although they remained alive and were in constant contact with the ME cells. Mammalian-cell damage occurred only when the organisms recovered pseudopodium function and began to migrate over the monolayers as they overcame the inhibitory effects of the antiserum. Cytochalasin B at a concentration of 10 microgram/ml, shown to prevent the engulfment of chick erythrocytes by amoebae, also inhibited the cytopathogenicity of Naegleria when incorporated in ME-cell culture medium. Despite repeated contact with active trophozoites, the ME cells showed only those morphological changes characteristically induced by cytochalasin B itself. The amoebae in turn showed signs of starvation after 3 or 4 days' incubation, suggesting that the feeding activity of trophozoites was suppressed. Colchicine, on the other hand, inhibited neither the ingestion of erythrocytes nor the destruction of ME cells by amoebae. It was concluded that the cytopathogenicity of N. fowleri in ME-cell cultures was due to physical rather than biochemical or cytotoxic mechanisms and was associated with the phagocytic activity of trophozoites.

Amoeba↗
PubMed1977

Cell size, macromolecular composition, and O2 consumption during agitated cultivation of Naegleria gruberi.

Cell size, macromolecular composition, carbohydrate utilization patterns, and O2 concentrations were measured throughout the growth stages of Naegleria gruberi in agitated culture in a complex medium. Biphasic logarithmic growth occurred during the intial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 X 10(6) amebae/ml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increase 31% to 35% per ameba at the end of each phase of log growth. DNA increased approximately 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O2 tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.

Amoeba↗
PubMed1979

Cellular and environmental variables determining numbers of flagella in temperature-shocked Naegleria.

Naegleria gruberi amebae normally transform into biflagellated cells. When subjected to high temperatures during flagellate differentiation, populations develop an average of 4-5 flagella/flagellate. Attempts to maximize this phenomenon by altering cellular and environmental variables revealed that: (a) few Naegleria isolates become multiflagellated: strain NB-1 gives the greatest response to heat shocks; (b) temperature is the most critical variable: highest numbers of flagella are obtained only if cells are temperature-shocked at precisely 38.2 +/- 0.1 C, then returned to 19-22 C to complete differentiation; (c) although pH alone does not affect numbers of flagella, a pH optimum of 5.5-7.0 exists for temperature-shocked cells; and (d) single cells in microdrops become multiflagellated, but the population response is density-dependent. Optimal conditions are described for growing, washing, and transforming amebae to generate reproducibly highest numbers of flagella.

Amoeba↗
PubMed1979

Occurrence of Naegleria and Acanthamoeba in aquaria.

Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi.

Amoeba↗
PubMed1978

Some further characteristics of the growth of Naegleria fowleri and N. gruberi in axenic culture.

The effects of pH, various viscosity of the medium, changed ratio between the concentrations of dissolved and corpuscular components in the medium, and dissolved inorganic salts on the growth of axenic cultures of Naegleria fowleri and N. gruberi have been studied. The cultures were grown in liquid CALYG and BCS media. The pH optimum was 6.5 for N. fowleri and 6.0--6.5 for N. gruberi. No negative influence on the growth of N. fowleri was observed even at 0.5% concentration of highly viscous methylcellulose, whereas the growth of N. gruberi was distinctly inhibited by more than 0.2% of methycellulose. N. fowleri preferred the osmotorphic and N. gruberi phagotrophic nutrition in the given system of cultivation. The growth of both Naegleria species was inhibited by 0.1 N concentration of sodium chloride and potassium chloride without any significant difference in the tolerance. The inhibitory effect of these salts correlated primarily with the concentration of chloride anion. The ability to grow in a medium with increased viscosity and the preference for osmotrophic nutrtion are, besides the higher temperature optimum determined earlier, further characteristics of the pathogenic species N. fowleri.

Amoeba↗
PubMed1979

Differences in virulence of Naegleria fowleri.

All pathogenic Naegleria fowleri isolated from the environment were highly virulent to mice when instilled intranasally. Axenic cultivation gradually decreased virulence of highly virulent strains. This decrease was most pronounced in environmental isolates and of minor importance in N. fowleri isolated from human cerebrospinal fluid. The low virulent strains obtained by continuous axenic cultivation appeared after clonation to consist of individuals with different virulence. Virulence could be enhanced in low virulent strains by brain passage and passages in Vero cell cultures, but could not be induced by these methods in nonvirulent strains isolated from the environment. Different mice strains showed different sensitivities to infection with pathogenic Naegleria. In addition, older mice were less sensitive than younger animals to low virulent strains.

Aging↗
PubMed1979

[Encephalitis due to Naegleria and Acanthamoeba. Comparison of organisms and diseases (author's transl)].

Naegleria and Acanthamoeba are ubiquitous, free-living amoebas. Infections with Naegleria are acquired nasally by exposure to water and are characterized by an acute fulminant hemorrhagic necrotizing meningoencephalitis leading to death. Acanthamoeba-infections occur in chronically ill, debilitated individuals. A patchy chronic or subacute granulomatous encephalitis is produced by hematogenous spread of the amoebas. The histological or clinical diagnosis is not difficult.

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↗
PubMed1979

Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.

Animals↗
PubMed1977

Ultrastructural observations of experimental Naegleria meningoencephalitis in mice: intranuclear inclusions in amebae and host cells.

Primary amebic meningoencephalitis was experimentallly produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality with average time of onset of symtoms of death occurring on the 7-8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.

Amebiasis↗
PubMed1979

In vitro susceptibilities of Naegleria fowleri strain HB-1 to selected antimicrobial agents, singly and in combination.

The overall prognosis of primary amoebic meningoencephalitis remains poor. The results of this study support previous finding that amphotericin B is the most efficacious drug against the Naegleria species in in vitro testing. In addition, the methyl ester of amphotericin B, a new derivative, also appears to be an effective agent. Of the drug combinations studied, amphotericin B plus minocycline and amphotericin B plus tetracycline showed synergy. The clinical significance of these findings remains to be determined.

Amoeba↗
PubMed1979

Susceptibility of Naegleria fowleri to delta 9-tetrahydrocannabinol.

Growth of the pathogenic amoeboflagellate Naegleria fowleri is inhibited by delta 9-tetrahydrocannabinol (delta 9-THC). delta 9-THC is amoebostatic at 5 to 50 micrograms/ml. delta 9-THC prevents enflagellation and encystment, but does not impair amoeboid movement. Calf serum at 10 and 20% (vol/vol) reduces the antiamoeba activity of delta 9-THC. Only 1-methoxy delta 8-tetrahydrocannabinol, of 17 cannabinoids tested, failed to inhibit growth of N. fowleri. Antinaeglerial activity was not markedly altered by opening the pyran ring, by converting the cyclohexyl ring to an aromatic ring, or by reversing the hydroxyl and pentyl groups on the benzene ring. delta 9-THC prevented the cytopathic effect of N. fowleri on African green monkey (Vero) cells and human epithelioma (HEp-2) cells in culture. delta 9-THC afforded modest protection to mice infected with N. fowleri.

Amebiasis↗