The Amoeba-to-Flagellate Transformation Test is not Reliable for the Diagnosis of the Genus Naegleria. Description of three new Naegleria spp.

Trophozoites of several isolates from one location in Australia have failed consistently to transform into flagellates, although they display all other characteristics of the genus Naegleria. When changing the standard transformation test, flagellates were produced. In phylogenetic trees derived from partial small subunit ribosomal DNA (SSUrDNA) sequences, one of these strains branches close to a cluster comprising N. clarki, N. australiensis, N. italica and N. jadini. It is proposed that these Australian isolates represent a new species, named N. fultoni (strain NG885). Failing to form flagellates since their isolation, even when different transformation procedures are used, are two Naegleria strains from Chile and Indonesia. In SSUrDNA-based phylogenetic trees the Chilean strain clusters with N. pussardi and the Indonesian strain clusters with N. galeacystis, but the degree of sequence difference from these described species (3.5% and 2.2%, respectively) is sufficient to propose that both of the strains represent new species, named N. chilensis (strain NG946) and N. indonesiensis (strain NG945), respectively. The close relationships between each of the new species and the Naegleria species with which they cluster in SSUrDNA-based trees were confirmed by ribosomal internal transcribed spacer region (ITS) sequence comparisxdons. In France, several non-flagellating N. fowleri strains were isolated from one location. ITS rDNA sequence comparisons indicated that they correspond to a ‘type’ of N. fowleri found in both Europe and the USA. A redefinition of the genus Naegleria is proposed as a consequence of these and previous findings.     

Naegleria lovaniensis new species: Isolation and identification of six thermophilic strains of a new species found in association with Naegleria fowleri

Six Naegleria strains, isolated previously in association with N. fowleri from thermal waters, were studied to further determine their antigenic relationship to N. fowleri and other Naegleria species. Results of immunofluorescent antibody and immunoelectrophoretic studies clearly established the antigenic divergence of the variants from N. fowleri, N. gruberi, and N. jadini. The variants were further distinguished from known Naegleria species by several ultrastructural characteristics, which included the complete enclosure of their nuclei with one or several layers of rough endoplasmic reticulum (RER) and extrusion of their nuclear material. Extruded nuclear material was observed in the cytoplasm completely sequestered by cisternae of the RER. The variants were also shown to be sensitive to agglutination induced by concanavalin A but not by wheat germ agglutinin. Based on the differences in the antigenicity, morphology, and lectin sensitivity between these six variants and established Naegleria species, we proposed that they should be established as a new species.     

Biochemical Identification and Phylogenetic Relationships in Free-Living Amoebas of the Genus Naegleria1

Using isoelectric focusing, the zymograms of 23 pathogenic and nonpathogenic Naegleria strains were studied for the activity of 16 enzymes. Certain enzymes (lactate dehydrogenase, L-threonine dehydrogenase, superoxide dismutase, acid phosphatase, malic enzyme, and leucine aminopeptidase) proved particularly useful from a practical point of view as they allow easy and reliable identification of pathogenic N. fowleri and N. australiensis as well as nonpathogenic N. lovaniensis strains. Genetic interpretation of these zymograms gave estimates of genetic distances that largely confirmed the taxonomic position of the Naegleria species. In addition, the genetic data suggest that there are two main phylogenetic groups in the genus Naegleria.     

Starch Gel Electrophoresis: An Effective Method for Separation of Pathogenic and Nonpathogenic Naegleria Strains*

SYNOPSIS. 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.     

Restriction Endonuclease Analysis of Mitochondrial DNA as an Aid in the Taxonomy of Naegleria and Vahlkampfia1

ABSTRACT Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

Morphology and Phylogenetic Analyses of Three Novel Naegleria Isolated from Freshwaters on Jeju Island,Korea, During the Winter Period

The genus Naegleria is one of the best known heterolobosean groups, and is the causative agent of primary amoebic meningoencephalitis. This group is rarely studied in temperate regions during winter. Here, three novel Naegleria were isolated from freshwaters on Jeju Island, Korea, during winter. Two isolates were amoeboflagellates, and one of the three amoebae did not undergo enflagellation. All amoebae had eruptive pseudopodia, and the layer of refractile granules around a large nucleus. They formed a cyst with ~2 pores in the cyst stage. The amoeboflagellate form had two flagella and no division in the flagellate stage, and no cytostome. These features are very similar to typical Naegleria. Furthermore, our isolates were able to grow at > 30 °C, suggesting that they had different thermophilicity from Naegleria in polar regions. All amoebae were largely encysted at 5 or 10 °C, indicating that they were likely encysted during winter. Based on the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequences, the phylogenetic analyses consistently revealed that the isolates are members of the Naegleria group. However, the isolates differ from other species in both phylogenetic trees. Thus, Naegleria in cold habitats appeared to have a high degree of novelty, but their thermophilicity may be dependent on locality.     

NACM, A Cytopathogenic Protein from Naegleria gruberi, EGs; Purification, Production of Monoclonal Antibody, and the Immunoidentification of a Product that Develops in NACM-treated Vertebrate Cell Cultures

The story of NACM involves the discovery of a deleterious response of cultured vertebrate cells to a component in cell-free lysates prepared from free-living amebae of the genus Naegleria; hence the acronym NACM derived from Naegleria ameba cytopathogenic material. The cellular reaction is the basis for the biological assay that has been fundamental in the study of the action of NACM in a variety of cell cultures. It also has been used in the determination of the physical characteristics, and to monitor the behavior of NACM during isolation procedures. All findings are compatable with the conclusion that NACM is a 35 Kd protein. Recently, the use of monoclonal antibodies (MAbs) prepared to amebae-derived purified NACM have resulted in visual display of a product that develops exclusively in NACM-treated cells. That cellular product is shown to be related to NACM by its immunostaining reaction with the MAb; the relationship of the MAb with NACM is demonstrated by its ability to neutralize the biological activity of NACM, and as an immunostain, to react with purified fractions of NACM and with whole amebae. The combination of these observations describes a unique set of interactions in which NACM, an amebic component, identified as a protein, has characteristics of an infectious agent when introduced into cultures of avian and mammalian cells.     

A Comparative Study of 14 Strains of Naegleria australiensis Demonstrates the Existence of a Highly Virulent Subspecies: N. australiensis italica n. spp.1

Fourteen strains of Naegleria australiensis, including the type strain, were compared for virulence for mice, maximum growth temperature, lectin agglutination, isoenzyme pattern, and total protein banding pattern. Their relation to other species of Naegleria also was compared by immunoelectrophoretic analysis. Strains with high virulence, comparable to that of N. fowleri, were found to be different in concanavalin A agglutination as well as with regard to zymograms and total protein patterns. Although serologically different from N. fowleri and reacting with N. australiensis antiserum in the fluorescent antibody test, these high-virulence strains differed in number of immunoelectrophoretic precipitin bands. Because of these results, the high-virulence strains are considered to be a subspecies of N. australiensis. The low-virulence strains showed minor differences from the type strain. Thus, N. australiensis does not appear to be as homogenous a species as N. fowleri. Pathogenic N. australiensis also seems to be more widespread than previously thought.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

Marinamoeba thermophila, a new marine heterolobosean amoeba growing at 50 °C

Two amoeba strains were isolated from marine sediment taken at the same place with 18 months interval from a region of the sea floor heated by extended submarine hot springs and fumaroles. These thermophilic amoebae grow at temperatures up to 50 °C. Sequences of the internal transcribed spacer demonstrated that the two strains belong to the same species and are different from any genus for which sequences are known. Phylogeny using small subunit ribosomal RNA places the amoeba in the Heterolobosea. Their closest relatives are the hypersaline flagellate Pleurostomum flabellatum and the hypersaline amoeba Tulamoeba peronaphora. The freshwater amoeboflagellate genera Naegleria and Willaertia belong to the same phylogenetic clade in the Vahlkampfiidae. The new marine species does not transform into flagellates. It forms cysts, which are round to ellipsoidal with few pores. Because of their unique place in the molecular phylogenetic tree, and because there is no morphologically identical species found in the literature, these isolates are considered to be a new species and a new genus, Marinamoeba thermophila.     

Phagocytic Activity of Three Naegleria Strains in the Presence of Erythrocytes of Various Types1

The phagocytic activities of N. lovaniensis (Aq/9/1/45D) and N. gruberi (1518/1f and 1518/1e) were studied in the presence of erythrocytes of various species: chicken, rabbit, goat, and human (A⁺, B⁺, and AB⁺ were tested). The percentage of amoebae with ingested red cells, the phagocytic index (PhI), can be considered as an expression of phagocytic activity. Under given conditions (erythrocyte concentration, incubation time, age of amoebic cultures) each strain of Naegleria prefers one erythrocyte type. Thus, for 72-h cultures, N. lovaniensis ingested more A⁺ type erythrocytes than did N. gruberi strains but had very low affinity for rabbit red cells except when very high concentrations were tested. Naegleria gruberi 1f was the most active of the three strains towards rabbit and B⁺ and AB⁺ human erythrocytes, but very low PhIs were obtained with goat erythrocytes. Naegleria gruberi le exhibited high phagocytic activity for every erythrocyte type except for rabbit red cells.     

Intranasal Immunization of Mice Against Naegleria fowleri1

ABSTRACT. The purpose of this research was to determine whether mice could be protected from lethal challenge with Naegleria fowleri by prior intranasal exposure to pathogenic and nonpathogenic Naegleria. Mortality ranged from 0 to 100% for mice inoculated intranasally (i.n.) with 5 × 10³ amebae of 13 human isolates of N. fowleri. Mice were immunized and challenged i.n. using live amebae of strains of low, medium, and high virulence. The greatest protection against lethal challenge was afforded by three immunizing doses of 10³ amebae per dose of the strain of medium virulence. Nonpathogenic N. gruberi also was used to immunize mice i.n. against lethal challenge with N. fowleri. Protection was greater following immunization with N. gruberi than it was after immunization with N. fowleri, suggesting that nonpathogenic N. gruberi may be a better immunogen in protecting mice against lethal naeglerial challenge.     

Identification of Ochratoxigenic Aspergillus Section Nigri Isolated from Grapes by ITS‐5.8S rDNA Sequencing Analysis and In Silico RFLP

To characterize Aspergillus section Nigri strains involved in the ochratoxin A (OTA) contamination of Tunisian wine and table grapes, a total of 33 strains were analysed. A molecular characterization of the isolates was performed by the amplification of internal transcribed spacer (ITS1‐5.8S rDNA‐ITS2) region combined with amplicon sequencing. Analysis of similarity between the obtained sequences and those deposited in the GenBank database was performed. Twelve strains were confirmed to belong to the Aspergillus carbonarius species. Strains belonging to the Aspergillus niger aggregate group were classified by in silico RFLP assay into two patterns N and T, corresponding to A. niger and Aspergillus tubingensis. Among the 21 OTA producing isolates analysed, 13 showed the T‐type pattern and 8 showed the N‐type pattern. The presented method showed to be a reliable alternative to the classic RFLP method. Our findings unambiguously revealed that multiple aspergilli species isolated from wine and table grape in Tunisia are able to produce OTA.     

Bottled Mineral Waters Polluted by Protozoa in Mexico

A survey of protozoa polluting bottled mineral water in Mexico was carried out using samples obtained from the three best-selling brands of bottled mineral water in the country. The organisms were concentrated through filtration procedures and subsequently cultured in sterile media. The cultures were observed over four weeks, with identification to the level of genus and species. Most commonly found were the amoebae Naegleria gruberi, Acanthamoeba astronyxis, and Vahlkampfia vahlkampfi (trophic as well as cystic stages) plus one flagellate, Bodomorpha minima. No ciliates were detected. The public health importance of the findings is obvious, since some strains of Naegleria and Acanthamoeba have the potential to cause human disease that may lead to death.     

Naegleria: A Research Partner For Cell and Developmental Biology1

ABSTRACT. Organisms in the genus Naegleria offer special opportunities for research in contemporary biology. the dramatic cell differentiation from amebae to flagellates is unique among eukaryotes in the rapidity, synchrony, reproducibility, homogeneity, and accessibility of a major phenotypic change. Environmental signals initiate a progressive signal transduction pathway in which genes are turned on, including those for several calcium-binding proteins, and newly synthesized proteins become localized in newly assembled organelles, including the centriole-like basal bodies, with the overall consequence that the cell changes its shape, motility, and behavior. This essay reviews research opportunities for which Naegleria excels, as well as interesting aspects of its biology that provide challenges for future investigations. Because these organisms alternate between two major eukaryotic motility forms, their phylogenetic position is also provocative. Although there are hints that Naegleria is capable of sexual reproduction in nature, mating has not yet been observed in the laboratory. In order to fully exploit the opportunities offered by this wonderful experimental system we are working to develop means to do genetic manipulation, in particular via DNA-mediated transformation.     

Detection of Biomarkers of Pathogenic Naegleria fowleri Through Mass Spectrometry and Proteomics

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix‐assisted laser‐desorption‐ionization‐time‐of‐flight mass spectrometry MALDI‐TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF‐TOF instrument. MALDI‐TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI‐TOF MS fingerprinting is a rapid, reproducible, high‐throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.     

Naegleria Actin Elicits Species-Specific Antibodies1

ABSTRACT. Actin, the major protein of amebae of Naegleria gruberi, proved to be strongly immunogenic in rabbits. The resulting precipitating antibodies are specific to actin of Naegleria. In a competitive solid-phase radioimmunoassay, these antibodies bound similarly to Naegleria G- and F-actin. Actins from amebae of Acanthamoeba and Dictyostelium, plasmodia of Physarum, sea urchin eggs, and vertebrate muscles gave no competition in the radioimmunoassay. Estimates of the amount of actin in Naegleria amebae ranged from a minimum of 5% of the total cell protein by radioimmunoassay to a maximum of 16% by electrophoresis. The unusual species specificity of these antibodies indicates that Naegleria actin, although conserved in many properties, is different enough to have unique antigenic determinants.     

Understanding the true burden of “Naegleria fowleri” (Vahlkampfiidae) in patients from Northern states of India: Source tracking and significance

The present study is an attempt to investigate the presence of Naegleria fowleri in Indian population. A total of 307 patients were enrolled and water samples were collected from both residential and surrounding areas of patients found positive for N. fowleri. The different species of Naegleria from both clinical and water samples were identified taxonomically. Recommended microbiological conventional techniques were used to identify different Naegleria stages and other free-living amoebae from the samples. PCR assays, using both genus and species specific primers were also optimized. None of the samples were positive by conventional microbiological examinations. However, PCR assays detected only three samples positive for N. fowleri. A total of 10 water bodies (ponds), that were used by Naegleria positive patients were examined. The pH and temperature of the water samples collected from water bodies ranged between 5.6–7.2 and 25–32 °C respectively. Among all the 10 water samples tested, four samples were positive for genus Naegleria by PCR assay, of which only two samples, showed positive amplification for N. fowleri. The sequence analysis of N. fowleri strain belonged to genotype II.     

Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing

Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas.     

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.