IgA and IgM anti-Naegleria fowleri antibodies in human serum and saliva

Antibodies from IgA and IgM classes that recognize Naegleria fowleri (Nf) proteins were detected by the ELISA assay in serum and saliva from three groups of people: (i) subjects with upper respiratory tract infections (URTI) living in the parasite-endemic area, (ii) healthy persons from the same area, and (iii) healthy persons from a parasite-nonendemic area. In serum and in saliva the titers of IgA antibodies to Naegleria fowleri in the group of patients with URTI was significantly higher than that of the healthy group in the parasite-endemic area; also the titers of IgM antibodies in serum were significantly higher in patients. On the contrary, in saliva the antibodies were higher in healthy people from the parasite-endemic area. In all cases the subjects from the parasite-nonendemic area had lower antibody titers in serum and saliva.     

Detection of Naegleria sp. in a thermal, acidic stream in Yellowstone National Park

An initial survey of sequences of PCR-amplified portions of the 18S rRNA genes from a community DNA clone library, prepared from an algal mat in a thermal, acidic stream in Yellowstone National Park, WY, USA, revealed among other sequences, several that matched Vahlkampfia. This finding prompted further investigation using primers specific for Naegleria. Sequences from a subsequent DNA clone library, prepared from the 5.8S rRNA gene and the adjacent internal transcribed spacer (ITS) regions of the rRNA, closely matched Naegleria and formed an independent lineage within a clade containing Naegleria sturti and Naegleria niuginiensis. The sequences may represent a new Naegleria species.     

Amoebacidal effects of serum from the American alligator (Alligator mississippiensis)

Treatment of axenic Naegleria gruberi cultures with alligator serum resulted in time-dependent amoebacidal activity, with measurable activity at 5 min and maximal activity occurring at 20 min. The amoebacidal activity was concentration dependent, with measurable activity at 25% serum, whereas treatment of amoebas with undiluted serum resulted in only 16% survival. The efficacy was dependent on the concentration of amoebas, with higher survival rates at high amoeba densities and lower survival rates at low amoeba densities. The amoeba-killing effects of alligator serum were broad in spectrum because the serum was effective against 3 strains of Naegleria species tested and 4 Acanthamoeba species, which have been reported to be resistant to human serum complement-mediated lysis. The amoebacidal effects of alligator serum were temperature dependent, with optimal activity at 15-30 C and a decrease in activity below 15 C and above 30 C. The amoebacidal activity of alligator serum was heat labile and protease sensitive, indicating the proteinaceous nature of the activity, and was also inhibited by ethylenediaminetetraacetic acid, which indicated a requirement for divalent metal ions. These characteristics strongly suggest that the amoebacidal properties of alligator serum are because of complement activity.     

Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake

Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.     

Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake.

Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.     

Modification of low density lipoprotein metabolism by growth factors in cultured vascular cells and human skin fibroblasts. Dependence upon duration of exposure

Phospholipase A, sphingomyelinase and lysophospholipase activities were examined in cell homogenates and cell-free culture media of virulent and virulent-attenuated Naegleria fowleri and nonpathogenic Naegleria gruberi. Homogenates of virulent N. fowleri contained from 3 to 250 times the lipolytic activity of virulent-attenuated and non-pathogenic Naegleria spp. Similarly, the cell-free media of virulent N. fowleri cultures contained large quantities of phospholipase A, lysophospholipase and sphingomyelinase while comparable activities in the cell-free media of virulent-attenuated and nonpathogenic Naegleria spp. were only slightly, if at all, detectable. Lipolytic enzymes accumulated in the media of virulent N. fowleri cultures at various stages during growth but not in virulent-attenuated and nonpathogenic Naegleria cultures. In general, phospholipase A and sphingomyelinase accumulated during the log phase of growth while lysophospholipase appeared only in the late stationary phase. We conclude that pathogenic Naegleria contain potent lipolytic enzymes that are released selectively into the media during growth. These enzymes could contribute to the pathogenesis of Naegleria-induced primary amoebic meningoencephalitis.     

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.     

Naegleria actin elicits species-specific antibodies

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.     

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.     

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.     

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.     

Loss of the ORF in the SSUrDNA group I intron of one Naegleria lineage.

We have found a Naegleria lineage in which the SSUrDNA contains a group I intron with a length of 375 nucleotides. This is a unique finding because all group I introns detected until now in Naegleria are 1.3 kilobases long and contain an open reading frame coding for 245 amino acids. Sequence data show that the 375 nucleotide-long intron is at the same place in the SSUrDNA as, and is descendant from, the 1.3 kilobase group I intron present in other species of Naegleria. Our data indicate that in one lineage of Naegleria the group I intron lost part of its DNA that is not contributing to the secondary structure but that carries the open reading frame. The amoeboflagellate genus Naegleria contains strains without the intron and strains with the intron, with or without an open reading frame. Therefore, this genus provides a unique opportunity to study the function and evolution of both the group I intron and the open reading frame.     

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.     

Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates.

Growth in an axenic medium composed by Chang (3rd Int. Congr. Parasitol. Munich Abstr. ICPIII 1:187-188, 1974) allowed separation of pathogenic from nonpathogenic Naegleria fowleri strains, since only the former show luxuriant growth in this medium. On the basis of these results, this medium was used in early screening for virulent Naegleria isolates. During an extensive ecological study, data were obtained on 102 Naegleria strains. Twenty of these strains grew luxuriantly in this liquid medium. Seventeen of them were tested by intranasal instillation in mice, and all proved to be highly pathogenic. Strains showing only moderate growth or no growth at all in this axenic medium were found to be nonpathogenic for mice. Moreover, it was found that using this medium in the early stage of Naegleria sampling favors isolation of pathogenic strains in mixtures of Naegleria. During these experiments, further evidence was obtained that thermal polluted waters are the main origin of N. fowleri in the environment.     

Thermal ecology of Naegleria fowleri from a power plant cooling reservoir

The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis. N. fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined. In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp. before and after thermal additions from a nuclear power plant. Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp. and pathogenic N. fowleri. Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis. N. fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir. The probability of isolating thermophilic Naegleria and pathogenic N. fowleri increased significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.     

Naegleria-Like Cysts In Cretaceous Amber From Central Kansas

ABSTRACT. Fossil protist cysts are reported from the mid-Cretaceous amber of Ellsworth County, Kansas, which is rich in terrestrial microfossils but contains no known macrofossils. On the basis of their distinctive morphology, the cysts can be referred to the genus Naegleria (Schizopyrenida); they most closely resemble cysts of the living species Naegleria gruberi. This is the first known fossil record for this group of amoebas. the current phylogenetic position and paleoecological role of Naegleria are discussed in relation to this find; it provides direct confirmation of morphological stasis in this group, which had previously been inferred from rRNA sequence divergence data.,     

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.     

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.     

Identification of Naegleria fowleri and other Naegleria spp. (free-living amoebae) using cellulose acetate membrane electrophoresis of glucose phosphate isomerase

Abstract A simple isoenzyme cellulose acetate membrane electrophoresis method with respect to glucose phosphate isomerase (GPI) was developed for the differentiation of the human pathogenic free-living amoeba Naegleria fowleri from other Naegleria spp. A single GPI band was detected in all the species tested, the relative mobility of which could be used to identify N. fowleri . Of the other Naegleria spp., only N. italica and N. jadini shared a common GPI mobility. No intraspecies variation in GPI profile was detected, regardless of whether the strains were cultured in monoxenic or axenic media. The technique is proposed as a useful means of identifying N. fowleri soon after isolation from the environment.