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.     

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

ABSTRACT: Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri , causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria , and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini , and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri , with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.     

High-Resolution Polyacrylamide Gradient Gel Electrophoresis (PGGE) of Isoenzymes from Five Naegleria Species

ABSTRACT. High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis , two N. lovaniensis , one N. jadini , two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis, two N. lovaniensis, one N. jadini, two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

Biochemical identification and phylogenetic relationships in free-living amoebas of the genus Naegleria

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.     

A comparative study of 14 strains of Naegleria australiensis demonstrates the existence of a highly virulent subspecies: N. australiensis italica n. spp

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.     

Isolation and identification of pathogenic Naegleria australiensis (Amoebida, Vahlkampfiidae) from a spa in northern Italy

Samples from therapeutic swimming pools and mud basins were cultured for free-living amoebae. Seven strains of pathogenic Naegleria species were isolated. Although some of the strains were as virulent as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis, they were identified as Naegleria australiensis with the indirect fluorescent-antibody technique. The virulence of the isolates for mice corresponded with the cytopathic effect for Vero cells. The N. australiensis strains were isolated from swimming pools with water temperatures ranging from 32 to 35 degrees C and from mud with temperatures from 25 to 43 degrees C. The presence of pathogenic N. australiensis in the swimming pools did not correlate with bacterial indicators.     

Isolation and identification of pathogenic Naegleria australiensis (Amoebida, Vahlkampfiidae) from a spa in northern Italy.

Samples from therapeutic swimming pools and mud basins were cultured for free-living amoebae. Seven strains of pathogenic Naegleria species were isolated. Although some of the strains were as virulent as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis, they were identified as Naegleria australiensis with the indirect fluorescent-antibody technique. The virulence of the isolates for mice corresponded with the cytopathic effect for Vero cells. The N. australiensis strains were isolated from swimming pools with water temperatures ranging from 32 to 35 degrees C and from mud with temperatures from 25 to 43 degrees C. The presence of pathogenic N. australiensis in the swimming pools did not correlate with bacterial indicators.     

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 (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 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 lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 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 ATCC 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.     

Discontinuous genetic variation among mesophilic Naegleria isolates: further evidence that N. gruberi is not a single species.

Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species--that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica. Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristics temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.     

Discontinuous Genetic Variation Among Mesophilic Naegleria Isolates: Further Evidence that N. gruberi Is Not a Single Species

Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species–that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica . Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristic temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.     

Molecular definition and the ubiquity of species in the genus Naegleria

To investigate the variability within species of the genus Naegleria, the ITS1,5.8S and ITS2 rDNA were sequenced of several strains of N. lovaniensis and its Western Australian variants, N. australiensis, N. fowleri, N. andersoni, N. jamiesoni, N. tihangensis, N. pringsheimi, N. pagei, N. gruberi sensu lato and a Naegleria lineage that lost a group I intron from the SSUrDNA twintron. As a result, it is possible to define a molecular species within the Naegleria genus. In addition, one strain of each different allozyme cluster was sequenced to investigate whether they belong to described species or should be treated as distinct new species. This leads to the proposal of eleven new species. The sequencing results from those Naegleria spp. of which several strains are available indicate that these species are ubiquitous. The only exception might be the species represented by the WA variants. However, there are still many Naegleria spp. for which only one strain has been isolated, hence, it is important that the search for more isolates should be continued worldwide.     

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.     

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.     

Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.     

Naegleria australiensis ssp. italica: experimental study in mice

A subspecies of Naegleria australiensis, N. australiensis italica, pathogenic for mice, was recently isolated and identified from an Italian thermal spa. We describe the histopathological changes of the central nervous system with experimental infection of albino mice. The histopathological patterns are intermediate to those seen with infection caused by N. fowleri and N. australiensis or Acanthamoeba spp. An acute inflammatory reaction was present within the choroid plexus, ependyma, midbrain, cerebellum, and basal ganglia. Occasional single amebic trophozoites were found within some microabscesses. Cysts were not identified. Involvement of the olfactory neuroepithelium and of the nasal mucosa was not detected.     

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.     

Naegleria fowleri and N. lovaniensis: differences in sensitivity to trimethoprim and other antifolates

The growth of Naegleria fowleri cultures in a BCS medium was not affected either by trimethoprim at 400 micrograms/ml or by aminopterine, 3,5-diaminopterine and methotrexate at 500 micrograms/ml. N. lovaniensis propagation in the same medium was inhibited with 10 micrograms/ml of trimethoprim, 50 micrograms/ml methotrexate and 100 micrograms/ml 3,5-diaminopteridine. Aminopterine was ineffective at a concentration of 500 micrograms/ml. The inhibitory effect of trimethoprim on N. lovaniensis cultures depended on the medium composition and could be neutralized by an addition of folic or tetrahydrofolic acids and a suspension of heat-killed Enterobacter aerogenes. Thymine, thymidine, hypoxantine and 2-amino-4-hydroxy-6-(tetrahydroxybutyl)-pteridine did not have an adverse effect. Trimethoprim activity in N. fowleri cultures could not be enhanced by the addition of Triton X-100 and Polymyxine B. Cryolyzate of N. fowleri amoebae did not influence the trimethoprim inhibition of N. lovaniensis cultures. Deviation in dihydrofolatereductase chemical structure or thymine dependency seems to be the probable explanation for N. fowleri antifolate resistance.     

Restriction endonuclease analysis of mitochondrial DNA as an aid in the taxonomy of Naegleria and Vahlkampfia

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.     

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.