Serology of Naegleria fowleri and Naegleria lovaniensis in a Hospital Survey1

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.     

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.     

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.     

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.     

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 throughput, real-time detection of Naegleria lovaniensis in natural river water using LED-illuminated Fountain Flow Cytometry

AIMS: To test Fountain Flow Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters. METHODS AND RESULTS: Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0.06 amoebae ml(-1), using a flow rate of 15 ml min(-1). CONCLUSIONS: This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.     

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.     

Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Abstract In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri , since Naegleriae ( N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini ) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, algae, y, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri .     

Resistance to Naegleria fowleri infection passively acquired from immunized splenocyte, serum or milk

A pathogenic free-living amoeba, Naegleria fowleri, causes primary amoebic meningoencephalitis to human and experimental animals. This infection is rare, but the mortality is very high. Nowadays, drug treatment or active immunization of human or mice are being tried with partial effectiveness. This study shows passive immunization effect by transfer of immunized spleen cells, serum, or milk from immunized mother in mouse experimental model. Young BALB/c mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri, and spleen cells and sera were collected for injection to recipient mice. There were seven transfer groups, i.e., immunized mouse serum, spleen cells, serum and spleen cells, normal mouse serum, spleen cells, serum and spleen cells, and control group. Three days later, BALB/c mice were inoculated with 1 x 10(4) trophozoites of N. fowleri intranasally. After infection, decreased mortality and prolonged survival time of mice were noted in immunized groups compared with non-immunized control group. The groups injected with immunized spleen cells or normal serum showed lower mortality than that of controls but showed no changes of serum IgG level. The groups injected with immunized serum or normal spleen cells showed increased serum IgG level after immunization but hundred percent mortality was observed. Mother mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri at the end of pregnancy and weaning period. Soon after the delivery, litters born of non-immunized mother were matched with immunized mother for feeding immune milk. After three weeks, the litters were infected with 1 X 10(4) trophozoites of N. fowleri or sacrificed for serum collection to measure the IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

The protective effects of monoclonal antibodies in mice from Naegleria fowleri infection]

Protective effects of monoclonal antibodies against N. fowleri were comparatively studied. BALB/c mice were treated with two types of monoclonal antibodies, Nf 2 and Nf 154, before and after the infection with N. fowleri. The mortality and mean survival times were then compared. Also, direct effect of the monoclonal antibodies on the N. fowleri trophozoites in vitro were observed. In vitro protective effects of the monoclonal antibodies were also studied in cells infected with N. fowleri. The observed results are summarized as follows: 1. Among mice pretreated twice before the infection with monoclonal antibody Nf 2(McAb Nf 2), only 15.8% were killed, and the mean survival time was 17.7 days. This was not much different from the mice pretreated once, as the mortality and mean survival time were 16.7% and 17 days. Those effects were compatible with monoclonal antibody Nf 154(McAb Nf 154). The above findings contrast with the mortality and mean survival time of the control mice, which were 22.7% and 14.6 days respectively. 2. Mice which received twice the McAb Nf 2 following N. fowleri infection incurred a 19.4% mortality rate with 13.6 days survival time; 17.9% and 15.8 days with on time administration, in contrast to the 25% and 14.6 days in the control group. 3. Marked agglutination effect of McAb Nf 2 or McAb Nf 154 were observed on N. fowleri trophozoites. 4. When N. fowleri trophozoites were treated with McAb Nf 2 or McAb Nf 154 combined with comments, the proliferation rate was more significantly suppressed than in that the control. 5. N. fowleri trophozoites treated with McAb Nf 2 or McAb Nf 154 showed an increased number of swollen mitochondria, disfigured cisternae, lipid droplets, and osmiophilic granules in the cytoplasm. 6. A remarkable protective effect of monoclonal antibodies was noticed in CHO cells infected with N. fowleri. More than 90.6% of the infected CHO cells survived, contrasted with 27% of untreated cells. The overall results in this study suggest that N. fowleri treated with monoclonal antibodies against N. fowleri reduce the mortality and prolong the survival time of the mice when the antibodies are administered before the infection. The protective effect of the monoclonal antibodies is surmised being caused by agglutination of the trophozoites.     

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.     

Acanthamoeba culbertsoni and Naegleria fowleri: occurrence of antibodies in man

Serum samples of 1,054 inhabitants of Bohemia (Czechoslovakia) were examined by means of indirect haemagglutination test with antigens from Naegleria fowleri and Acanthamoeba culbertsoni. With N. fowleri antigen the frequency of positive reactions did not exceed 3.5 per cent in lowest serum dilutions only. N. fowleri could not be identified as a possible causative agent in any chronic form of disease in man. Significant accumulation of positive findings with A. culbertsoni antigen was observed in hepatitis A patients and convalescents (52 per cent). The potential interpretations of this phenomenon are discussed.     

The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis

From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.     

Chemotaxis by Naegleria fowleri for bacteria

Naegleria fowleri amebae demonstrated a chemotactic and chemokinetic response toward live cells and extracts of Escherichia coli and other bacterial species when experiments were performed using a blind-well chemotaxis chamber. The peptide N-formyl-methionyl-leucyl-phenylalanine acted as a chemokinetic rather than a chemotactic factor for N. fowleri amebae. Competition experiments in which nerve cell extracts or bacteria were placed on either side of the filter in chemotaxis chambers resulted in increased movement towards bacteria. A scanning electron microscopy study of the interaction of N. fowleri with different bacterial species confirmed that when the amebae were near ingestible bacteria they moved toward the bacteria by pseudopod formation. Naegleria fowleri appeared to respond to bacteria by three interrelated but distinct processes: chemokinesis, chemotaxis, and formation of food cups.     

Calcium-dependent protection from complement lysis in Naegleria fowleri amebae

Pathogenic Naegleria fowleri amebae are resistant to the lytic effects of serum complement. The presence of surface glycoproteins or removal of the membrane attack complex (MAC) of complement from the cell surface by vesiculation serve to protect the amebae from complement lysis. The specific mediators important in stimulating complement resistance are not defined. These studies were undertaken to examine the effect of Ca(2+) ions in initiating complement resistance of N. fowleri in contrast to non-pathogenic complement-sensitive N. gruberi. Chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) or chelation of intracellular calcium with 1,2-bis-(O-Aminophenoxy) ethane-N,N,N,N tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) increased complement lysis of N. fowleri. Chelation of calcium ions did not affect complement sensitivity of N. gruberi. Increased lysis of ionomycin-treated N. fowleri was detected after exposure to serum complement, suggesting that a threshold level of Ca(2+) mediates complement resistance before survival mechanisms are overwhelmed and lysis occurs. A differential influx of Ca(2+) ions occurred in fura-2 labeled N. fowleri after deposition of complement component C9 to form the MAC complex on the cell surface in comparison to N. gruberi. These studies suggest that Ca(2+) ions influence complement resistance in N. fowleri but do not play a role in altering the sensitivity of N. gruberi to complement.     

Isolation of a unique membrane protein from Naegleria fowleri

Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.