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.     

Isoenzyme and Total Protein Analysis by Agarose Isoelectric Focusing, and Taxonomy of the Genus Acanthamoeba

Thirty axenically grown reference strains belonging to 15 different Acanthamoeba spp. were investigated for isoenzyme patterns by agarose isoelectric focusing in the pH range 3–10. Zymograms of acid phosphatase, leucine amino peptidase, malate dehydrogenase, propionyl esterase, glucose phosphate isomerase, phosphoglucomutase, and alcohol dehydrogenase were compared. The same strains were also analyzed for protein patterns separated by agarose isoelectric focusing in a pH gradient of 5–8. The results suggested changes in taxonomy within morphology group II of Pussard & Pons. Acanthamoeba paradivionensis becomes a synonym of A. divionensis. Although this species seems to be related to A. rhysodes, it could not be concluded that the species names are synonyms since the type strain of A. rhysodes was not available for comparison. In the subgroup A. polyphaga–A. quina–A. lugdunensis, A. lugdunensis becomes the species name for pathogenic strains of this subroup, A. quina for the nonpathogenic strains, while A. polyphaga is the species name for an atypical strain. Two strains of A. castellanii showed different zymograms from strain Neff of this species, but related protein patterns. In group III, A. pustulosa is found to be a synonym of A. palestinensis, while one strain of A. lenticulata is also found to belong to the A. palestinensis species. All other species names in both morphology groups could be retained as valuable, on the basis of the techniques used. Group I was not investigated, as axenic cultures could not be obtained.     

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.     

Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.     

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.     

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.     

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.     

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.     

Population Structure in Naegleria fowleri as Revealed by Microsatellite Markers

Naegleria sp. is a free living amoeba belonging to the Heterolobosea class. Over 40 species of Naegleria were identified and recovered worldwide in different habitats such as swimming pools, freshwater lakes, soil or dust. Among them, N. fowleri, is a human pathogen responsible for primary amoeboic meningoencephalitis (PAM). Around 300 cases were reported in 40 years worldwide but PAM is a fatal disease of the central nervous system with only 5% survival of infected patients. Since both pathogenic and non pathogenic species were encountered in the environment, detection and dispersal mode are crucial points in the fight against this pathogenic agent. Previous studies on identification and genotyping of N. fowleri strains were focused on RAPD analysis and on ITS sequencing and identified 5 variants: euro-american, south pacific, widespread, cattenom and chooz. Microsatellites are powerful markers in population genetics with broad spectrum of applications (such as paternity test, fingerprinting, genetic mapping or genetic structure analysis). They are characterized by a high degree of length polymorphism. The aim of this study was to genotype N. fowleri strains using microsatellites markers in order to track this population and to better understand its evolution. Six microsatellite loci and 47 strains from different geographical origins were used for this analysis. The microsatellite markers revealed a level of discrimination higher than any other marker used until now, enabling the identification of seven genetic groups, included in the five main genetic groups based on the previous RAPD and ITS analyses. This analysis also allowed us to go further in identifying private alleles highlighting intra-group variability. A better identification of the N. fowleri isolates could be done with this type of analysis and could allow a better tracking of the clinical and environmental N. fowleri strains.     

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts. International journal for Parasitology4: 139–142. Cysts of 4 strains of non-pathogenic Naegleria gruberi and 5 strains of pathogenic Naegleria fowleri were examined in the scanning electron microscope. Excystment of the Naegleria gruberi amoebae occurred via preformed exit pores in the cyst wall. Similar structures were not found in the cysts of Naegleria fowleri, and excystment occurred by rupture of the cyst wall. The sequence of cyst wall rupture is illustrated for one of the pathogenic strains.     

Movement of Naegleria fowleri Stimulated by Mammalian Cells In Vitro1

ABSTRACT. 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.     

Naegleria fowleri and Naegleria gruberi 20S proteasome: identification and characterization

The Naegleria are ubiquitous free-living amoebae and are characterized by the presence of three phases in their biological cycle: trophozoite, cyst and flagellate. Of this genus, only Naegleria fowleri has been reported as pathogenic to humans. The proteasome is a multi-catalytic complex and is considered to be the most important structure responsible for the degradation of intracellular proteins. This structure is related to the maintenance of cellular homeostasis and, in pathogenic microorganisms, to the modulation of their virulence. Until now, the proteasome and its function have not been described for the Naegleria genus. In the current study, using bioinformatic analysis, protein sequences homologous to those reported for the subunits of the 20S proteasome in other organisms were found, and virtual modelling was used to determine their three-dimensional structure. The presence of structural and catalytic subunits of the 20S proteasome was detected by Western and dot blot assays. Its localization was observed by immunofluorescence microscopy to be mainly in the cytoplasm, and a leading role of the chymotrypsin-like catalytic activity was determined using fluorogenic peptidase assays and specific proteasome inhibitors. Finally, the role of the 20S proteasome in the proliferation and differentiation of Naegleria genus trophozoites was demonstrated.     

Acid-active neuraminidases in the growht media from cultures of pathogenic Naegleria fowleri and in sonicates of rabbit alveolar macrophages

Using bovine mucin and isolated human myelin as source of sialic acid, we demonstrate the presence of neuraminidase activities in the growth media of pathogenic, but not nonpathogenic, Naegleria sp. and in sonicates of rabbit alveolar macrophages. Neuraminidase activity was maximal at pH 4.5 and 5.0, and the specific activity for sialic release was up to 13-fold greater with mucin that with human myelin. Activity in the growth media from cultures of pathogenic Naegleria fowleri was ion-independent, while that of macrophage sonicates required divalent cation; optimal activity was noted with 2.5 mM Zn², while Mg²⁺ and Mn²⁺ supported activity to a lesser extent. Such acid-active neuraminidases may contribute to the reported glycolipid alterations associated with the demyelinating diseases.     

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.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

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.     

Concentration of Naegleria fowleri in natural waters used for recreational purposes in Sonora, Mexico (November 2007-October 2008)

A survey was designed to know the concentration of Naegleria fowleri in recreational areas in Hornos, Sonora, during a year. Samples were taken monthly at La Isleta and Las Palmas and the total amoeba counts were obtained by the most probable number method (MPN). The identification of N. fowleri was made by PCR. The maximum concentration of total thermophilic amoebae was 9175 MPN/L for La Isleta and 3477 MPN/L for Las Palmas. Thermophilic Naegleria were present mainly during summer and fall. October’s concentrations were up to 201 MPN/L, at both places. The maximum concentrations of N. fowleri were 201 MPN/L and 18 MPN/L for La Isleta and Las Palmas respectively, and were isolated from August to October. The presence of N. fowleri in these particular natural bodies of water reinforces the need for adaptation of preventive measures to avoid cases of primary amoebic meningoencephalitis.     

Enzyme activities in the plasma of rainbow trout, Salmo gairdneri Richardson; the effects of nutritional status and salinity

The levels of lactate dehydrogenase, hydroxy butyric dehydrogenase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, glutamate dehydrogenase, alkaline phosphatase and leucine amino peptidase were determined in the plasma of rainbow trout. The protein concentration and the amount of alkaline phosphatase were reduced in starving trout. Fed trout showed reduced lactate dehydrogenase activity. There was a significant correlation between the condition factor, most of the enzyme activities and the protein concentration. At 10 parts per thousand salinity the activities of alkaline phosphatase and leucine amino peptidase were significantly elevated, while lactate dehydrogenase activity was significantly decreased.     

Biochemical properties of latices from the Euphorbiaceae

Latex sera from 18 Euphorbia species were surveyed for protein, carbohydrate and total solid contents. Protease, esterase, alkaline and acid phosphatase, N-acetyl-β-glucosaminidase, α-mannosidase, α- and β-galactosidase, α- and β-glucosidase, α-amylase, lysozyme, leucine amino peptidase and cellulase activities were also measured. The effects of nine protease inhibitors were assayed as were haemagglutinating (lectin) contents. Two-dimensional (isoelectric focusing and SDS) PAGE maps of the sera were made. The results obtained show that the latices have widely varying biochemical properties.