Naegleria Species Research Articles

Genome Structure and Evolution of Naegleria and its Relatives

In the 4 yr since the molecular biology of DNA in Naegleria was last reviewed several major advances have been made, and these are reviewed here: isolation and characterization of mitochondrial and ribosomal DNAs; enumeration of chromosomal DNAs by pulsed field gel electrophoresis; sequence analysis of differentially expressed genes; phylogenetic placement of the genus Naegleria among the eukaryotes and Naegleria species within the genus.

SrRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda).

A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent.

Application of isoenzymatic typing to the identification of nonaxenic strains of Naegleria (Protozoa, Rhizopoda).

Isoenzymatic typing of the different species of Naegleria was studied by comparing isoelectric focusing on axenic and nonaxenic strains using the most discriminating enzymes: lactate dehydrogenase (LDH), malic enzyme (ME), beta-hydroxybutyrate dehydrogenase (beta-HBDH), superoxide dismutase (SOD), and acid phosphatase (AP). The results show that bacteria in nonaxenic cultures have no influence on the interpretation of zymograms. The specificity of the enzymatic patterns of each strain is maintained in all of these enzymatic systems except LDH. With SOD, the existence of a supplementary isoenzyme among all of the nonaxenic strains is probably linked to an enzymatic induction phenomenon.

Naegleria andersoni n.sp., a cosmopolitan amoebo-flagellate, with two subspecies

A new Naegleria species, N. andersoni, is described that occurs world-wide. The new species consists of two subspecies and can be distinguished from previously described species on the basis of antigenic, isoenzyme and restriction enzyme DNA fragment differences and in maximum growth temperature tolerated. N. andersoni is not pathogenic in experimental animals and shows no cytopathic effect in cell culture. The electrophoretic karyotype differs between strains of the species, and most of all between the two subspecies, N. a. andersoni and N. a. jamiesoni.

Production of monoclonal antibodies to Naegleria fowleri, agent of primary amebic meningoencephalitis

Monoclonal antibodies (MAbs) to Naegleria fowleri, the etiologic agent of primary amebic meningoencephalitis (PAM), have been produced and used as probes to identify N. fowleri amebae in brain sections of patients who died of that disease. These MAbs were characterized for their specificity by the indirect immunofluorescence assay (IIF), dot immunobinding assay (DIBA), and enzyme-linked immunotransfer blot technique (EITB). The MAbs reacted intensely with all strains of N. fowleri tested originating from different geographic areas in the IIF and DIBA tests, but showed no reactivity with four other species of Naegleria, N. gruberi, N. jadini, N. lovaniensis, and N. australiensis, or a strain of Acanthamoeba castellanii. In the EITB assay the MAbs reacted with the antigens of N. fowleri and produced intensely staining bands at the 160-, 104-, 93-, and 66-kilodalton (kDa) regions and several minor bands at the 30- and 50-kDa regions. The MAbs also reacted with the antigens of N. lovaniensis and produced a darkly staining band at 160 kDa and a diffusely staining band at 116 kDa, indicating that these antigens were shared by the two species. The MAbs, however, showed no reactivity with N. jadini and N. gruberi in the EITB assay.

Specificity of antibodies from human sera for Naegleria species

Serum samples from adult humans in North Carolina and Pennsylvania were assayed for antibodies against four Naegleria species: N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis. Agglutinating activities of serum samples from North Carolina subjects were higher for N. fowleri than were those from Pennsylvania subjects. The distributions of agglutination titers of human serum samples for N. australiensis, N. gruberi, and N. lovaniensis were heterogeneous. The agglutination capabilities of selected serum samples absorbed with rounded, killed trophozoites of N. australiensis and N. lovaniensis were distinctly different, as were those of serum samples absorbed with N. fowleri and N. gruberi. N. australiensis and N. gruberi shared some agglutinating antigens, as did N. fowleri and N. lovaniensis. The agglutinating activities of most serum samples correlated with the capability of their immunoglobulin M (IgM) to bind to antigens in extracts of Naegleria species but not with the capabilities of their IgG to bind to antigens of Naegleria species. Absorption of IgM binding capability with rounded, killed trophozoites established that N. gruberi was distinctly different from N. fowleri and N. lovaniensis but that N. fowleri and N. lovaniensis shared surface antigens. The proteins in extracts of the four Naegleria species were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tested for their ability to bind immunoglobulins in a serum sample. The antigens of the four species that bound IgM or IgG in the tested serum sample were separated by SDS-PAGE, and when they were incubated with anti-IgM or anti-IgG, they gave distinct profiles. There was one distinct, shared antigen that had a molecular size of 40,000 daltons. Absorption of the test serum with killed, rounded trophozoites did not markedly change the immunoglobulin binding profile for Naegleria internal antigens separated by SDS-PAGE and did not remove the shared 40,000-dalton protein(s). These results demonstrate that the four Naegleria species have antigenically distinct surfaces and that humans have been individually exposed to antigens of Naegleria species.

Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoeba Naegleria lovaniensis and Naegleria gruberi.

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniesis, it is accompanied by lanosterol and parkeol, as well as by the 24,25-dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4 alpha-methylsterols which are present in similar amounts as those of 4,4-desmethylsterols (3-5 mg/g, dry weight). 4 alpha-Methylergosta-7,22-dienol was identified as a new compound. Ergosterol was the major 4,4-desmethylsterol, accompanied by small amounts of C27 and other C28 sterols. Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4 alpha-methyl-delta 7-sterols and the appearance of the unusual delta 6,8,22-ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the delta 8----delta 7 isomerase, the small amounts of delta 7-sterols formed being converted into ergosterol which is still present in fenpropimorph-exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1-14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3-3H]squalene epoxide was efficiently cyclized by a cell-free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non-photosynthetic phyla (animal and fungi) and parkeol are more likely dead-ends of this biosynthetic pathway. The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine-like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics.(ABSTRACT TRUNCATED AT 400 WORDS)

First Evidence for Diploidy and Genetic Recombination in Free-Living Amoebae of the Genus Naegleria on the Basis of Electrophoretic Variation

Electrophoretic variation for 15 enzyme-coding genes was studied in various Naegleria (Rhizopoda, Vahlkampfiidae) species. The occurrence of complex banding patterns provided the first evidence of a diploid structure of the genome of these amoebae. The putative loci identified were found not to be linked and the genotypic distribution suggested chromosomal recombination for one species (Naegleria lovaniensis).

Antiamebic activity of new acridinic derivatives against Naegleria and Acanthamoeba species in vitro.

In vitro antiamebic activity of selected acridine derivatives has been investigated against Naegleria and Acanthamoeba species. The most active compounds belong to the 9-thioacridanone and the 1,2,3,4-tetrahydro-9-thioacridanone series. In addition, some structure-activity relationships are proposed.

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.

Movement of Naegleria fowleri stimulated by mammalian cells in vitro.

Naegleria fowleri amebae, but not those of N. australiensis, N. gruberi, or N. lovaniensis, demonstrated enhanced motility when placed in proximity to mammalian cells. Amebae of nonpathogenic species of Naegleria, however, were more motile in cell culture medium than the amebae of N. fowleri. The locomotory response of highly pathogenic mouse-passaged N. fowleri amebae to nerve cells was greater than axenically cultured amebae. The enhanced mobility elicited by whole nerve cells or disrupted nerve cells was not directed migration but chemokinetic. Naegleria fowleri responded to disrupted neuroblastoma cells more vigorously than to disrupted African green monkey kidney (Vero) cells.

Migration patterns of pathogenic and nonpathogenic Naegleria spp.

Four species of Naegleria were tested for their ability to migrate under agarose. Pathogenic N. fowleri strains exhibited rapid locomotion at 37 degrees C. Environmental isolates of N. fowleri moved faster than clinical isolates which had been kept in axenic culture for longer periods, and this result was confirmed by using the 84-2205-7 strain kept in axenic culture for 1 or 5 months. Nonpathogenic N. gruberi strains migrated actively at 28 degrees C but not at 37 degrees C; moreover, even at 28 degrees C, active amoebae constituted only a small proportion of the whole. The temperature-tolerant, nonpathogenic species N. lovaniensis moved more slowly than N. fowleri at 37 degrees C. In contrast, N. australiensis, which is temperature tolerant as well as pathogenic for mice, migrated at a rate comparable to that of N. fowleri. There appears to be a direct correlation between the locomotive ability of free-living amoebae and their pathogenic potential.

Rapid identification of thermophilic Naegleria, including Naegleria fowleri using API ZYM system.

The suitability of the API ZYM system for identifying thermophilic Naegleria species, based on enzyme presence and activity, was investigated. Replicate testing on strains of N fowleri, N lovaniensis, and N australiensis cultured in a monoxenic and an axenic medium showed that the system could provide a rapid and reproducible means of identifying the species soon after primary isolation. No single enzyme was found specific for any one species, but considerable differences were found in the patterns of activity of acid phosphatase and leucine arylamidase. When these were compared the species could be differentiated. Use of the system in conjunction with a simple culture method is proposed as a readily available means of monitoring environmental and public bathing sites to prevent primary amoebic meningoencephalitis.

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.

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.

Erster Nachweis einer pathogenenNaegleria-Art (N. australiensis De Jonckheere 1981) in Deutschland

For the first time three strains of the newly described species of pathogenic Naegleria,N. australiensis De Jonckheere 1981 were isolated from a thermally polluted canal in Western Germany. They were pathogenic for mice with a lower mortality and a longer incubation period compared to mice infected withN. fowleri (Carter 1970). By the immunofluorescence-method these strains could be identified asN. australiensis.

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.

The nature of a cytopathogenic material present in amebae of the genus Naegleria.

The Naegleria ameba cytopathogenic material (NACM) obtained from free-living and/or pathogenic amebae has been partially isolated and characterized. The evidence indicates that the material is a protein or has a protein component, and is in the estimated molecular weight range of 50,000 daltons. The partially isolated NACM, like its counterpart in the amebic lysates, is capable of sustaining itself in tissue cultures at a temperature which otherwise would inactivate it, and in serial passages through multiple dilutions. No distinctions between the NACMs from amebae of different Naegleria species or strains have been found in the biological response to the various treatments studied.

Diagnostic review of Derrick's case of generalized amoebiasis (? Iodamoeba butschlii)

In 1943, the late E. H. Derrick performed an autopsy in Brisbane on a case of generalized amoebiasis (stomach, small intestine, caecum, ascending and transverse colon, gastric and mesenteric lymph nodes, lungs, brain) in a Japanese prisoner-of-war who had been captured at Buna in New Guinea. The causative amoeba was obviously not Entamoeba histolytica and, because of its ‘limax’ type of nucleus, was thought to be Iodamoeba butschlii or an amoeba closely resembling it. Iodamoeba butschlii is considered to be non-pathogenic. Furthermore, the amoebae and pathology in sections of brain in human primary meningo-encephalitis due to Naegleria fowleri (and transferred infection in mice) appeared identical with those in Derrick's case. On these counts it was decided to attempt immunofluorescent diagnosis on the material which was available. Sections cut from original paraffin blocked material (brain) were subjected to immunofluorescence by W. P. Stamm in London in 1975 with antisera prepared from species of Naegleria, Hartmannella, and Acanthamoeba. Tabled 1 Antiserum Titre Ami HB1 (N. fowleri) 1:32 Anti N. gruberi 0 Anti HN3 (H. rhysodes) 0 Anti Ryan (H. rhysodes) 0 Anti A1 (A. culbertsoni) 0 Anti Entamoeba histolytica 0 Open table in a new tab Sections of lung and colon gave the same titre (1:32) to anti HB1 (N. fowleri) antiserum in 1976. Although there is immunofluorescent antigenic overlap between strains of Hartmannellae (Hartmannella and Acanthamoeba), there is virtually no such overlap between the Naegleriae and Hartmannellae or between nonpathogenic (e.g., N. gruberi) and pathogenic (e.g. N. fowleri) strains of Naegleria. In view of the age of the material tested (33 years), it is considered that the titre obtained to anti HB1 (N. fowleri) is significant. Accordingly, it is submitted that the diagnosis in this case should now be one of generalized amoebiasis due to N. fowleri or a similar pathogenic species. In 1943, the late E. H. Derrick performed an autopsy in Brisbane on a case of generalized amoebiasis (stomach, small intestine, caecum, ascending and transverse colon, gastric and mesenteric lymph nodes, lungs, brain) in a Japanese prisoner-of-war who had been captured at Buna in New Guinea. The causative amoeba was obviously not Entamoeba histolytica and, because of its ‘limax’ type of nucleus, was thought to be Iodamoeba butschlii or an amoeba closely resembling it. Iodamoeba butschlii is considered to be non-pathogenic. Furthermore, the amoebae and pathology in sections of brain in human primary meningo-encephalitis due to Naegleria fowleri (and transferred infection in mice) appeared identical with those in Derrick's case. On these counts it was decided to attempt immunofluorescent diagnosis on the material which was available. Sections cut from original paraffin blocked material (brain) were subjected to immunofluorescence by W. P. Stamm in London in 1975 with antisera prepared from species of Naegleria, Hartmannella, and Acanthamoeba. Sections of lung and colon gave the same titre (1:32) to anti HB1 (N. fowleri) antiserum in 1976. Although there is immunofluorescent antigenic overlap between strains of Hartmannellae (Hartmannella and Acanthamoeba), there is virtually no such overlap between the Naegleriae and Hartmannellae or between nonpathogenic (e.g., N. gruberi) and pathogenic (e.g. N. fowleri) strains of Naegleria. In view of the age of the material tested (33 years), it is considered that the titre obtained to anti HB1 (N. fowleri) is significant. Accordingly, it is submitted that the diagnosis in this case should now be one of generalized amoebiasis due to N. fowleri or a similar pathogenic species.

Freeze Preservation of Pathogenic and Nonpathogenic Naegleria Species

Freeze Preservation of Pathogenic and Nonpathogenic Naegleria Species