Pathogenic Naegleria Fowleri Research Articles

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Nanoparticle-Terpene Fusion: A Game-Changer in Combating Primary Amoebic Meningoencephalitis Caused by Naegleria fowleri.

Pathogenic Naegleria fowleri (N. fowleri) are opportunistic free-living amoebae and are the causative agents of a very rare but severe brain infection called primary amoebic meningoencephalitis (PAM). The fatality rate of PAM in reported cases is more than 95%. Most of the drugs used againstN. fowleri infections are repurposed drugs. Therefore, a large number of compounds have been tested againstN. fowleri in vitro, but most of the tested compounds showed high toxicity and an inability to cross the blood-brain barrier. Andrographolide, forskolin, and borneol are important natural compounds that have shown various valuable biological properties. In the present study, the nanoconjugates (AND-AgNPs, BOR-AgNPs, and FOR-AgNPs) of these compounds were synthesized and assessed against both stages (trophozoite and cyst) ofN. fowleri for their antiamoebic and cysticidal potential in vitro. In addition, cytotoxicity and host cell pathogenicity were also evaluated in vitro. FOR-AgNPs were the most potent nanoconjugate and showed potent antiamoebic activity againstN. fowleriwith an IC50 of 26.35 μM. Nanoconjugates FOR-AgNPs, BOR-AgNPs, and AND-AgNPs also significantly inhibit the viability of N. fowleri cysts. Cytotoxicity assessment showed that these nanoconjugates caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 μg/mL, while also effectively reducing the cytopathogenicity of N. fowleri trophozoites to the HaCaT cells. The outcomes of our experiments have unveiled substantial potential for AND-AgNPs, BOR-AgNPs, and FOR-AgNPs in the realm of developing innovative alternative therapeutic agents to combat infections caused by N. fowleri. This study represents a significant step forward in the pursuit of advanced strategies for managing such amoebic infections, laying the foundation for the development of novel and more effective therapeutic modalities in the fight against free-living amoebae.

Molecular characterization and phylogenetic analysis of FLA from different water sources in Egypt

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. In addition to their natural distribution, some species have been documented as pathogenic to humans. The main aim of the current study was the molecular identification, sequencing and phylogenetic analysis of morphologically detected FLA in water sources in El-Qalyubia, Egypt. A total of 96 water samples were collected from different water sources. Each water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive FLA were subjected to PCR, PCR products were sequenced and the obtained sequences were phylogenetically analysed. FLA were found in 41 water samples examined (42.7%). Nile water and groundwater were the sources with the highest prevalence rates (83.3 and 62.5%, respectively). Naegleria italica was first identified in Egypt from the waters of the Nile. In addition, Vahlkampfia spp. and Hartmannella spp. were also detected. However, other FLA species, including Acanthamoeba spp. and the pathogenic Naegleria fowleri, previously reported in Egypt, were not included in this study. The recent identification of these FLA in the Egyptian waters related to human populations indicates the need for more phylogenetic studies using larger sample sizes to investigate their potential threat to human health.

Effect of a mannose to the interactions between Naegleria fowleri and pathogenic bacteria

Introduction and Aim: In this study, the interaction between pathogenic Naegleria fowleri and pathogenic bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis and Salmonella typhi was analyzed by a monosaccharide of mannose. Moreover, since the form of N. fowleri was found in diseases as cysts, the interaction between cysts and bacteria was analyzed. Materials and Methods: In order to analyze the role of a monosaccharide called mannose in bacterial interaction, the analysis of bacterial association, invasion, and survival for amoeba treated with mannose was performed. N. fowleri trophozoites or cysts were pre-treated with a mannose at a concentration of 10, 50 and 100 mM for 1 hr at 37°C. Results: The MRSA association was hardly suppressed until the concentration of mannose was 50 mM, but its association was reduced by about 168% to N. fowleri trophozoites by 100 mM mannose. Compared to the results for MRSA, the association of E. faecalis had little effect by mannose. Very interestingly, although S. typhi showed much higher invasion than the above MRSA and E. faecalis, it did not survive at all within N. fowleri trophozoites. Ten mM mannose showed a nearly similar 1% association with N. fowleri cyst treated, but not with 50 mM and 100 mM mannose treated N. fowleri cyst at all. Conclusion: The association and invasion of S. typhi was highest for N. fowleri trophozoites and cysts, but the three bacteria did not survive in N. fowleri trophozoites and cysts.

First identification of Naegleria species and Vahlkampfia ciguana in Nile water, Cairo, Egypt: Seasonal morphology and phylogenetic analysis.

In Egypt, there is a scarcity of data concerning Naegleria (N.) family, with a shortage of phylogenetic studies. This study's aim was molecular detection, sequencing and phylogenetic analysis of morphologically identified Nagleria and to determine natural seasonal distribution of Nagleria species in water sources of Greater Cairo, Egypt. A total of 120 water samples were collected during each season over a year. Every water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive Nagleria-like isolates were subjected to Nagleria genus and species-specific PCR targeting rDNA gene, PCR products were sequenced and obtained sequences were phylogenetic analyzed. Nile River water was the only source found to contained Naegleria. For the first time in Egypt, Vahlkampfia ciguana and the Naegleria species N.australiensis, N.philippinensis and N.neojejuensis were identified from the Nile water. The pathogenic Naegleria fowleri, previously reported in Egypt, was however not detected in this study. Interestingly, there were no seasonal variations in prevalence of Naegleria spp.; yet, there was seasonal diversity in the water samples of the same site. These newly discovered Vahlkampfiidae in Egyptian aquatic environments indicate the need for further phylogenetic investigations using bigger sample sizes in order to determine their potential risk for human health.

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae.

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page’s amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri

Naegleria fowleri, known as the brain-eating amoeba, causes acute primary amoebic meningoencephalitis. During swimming and other recreational water activities, N. fowleri trophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due to N. fowleri infection, human macrophage cells (THP-1 cells) were cocultured with N. fowleri trophozoites in a noncontact system, and consequently, interleukin-1β (IL-1β) production was estimated. Caspase-1 activation and IL-1β production from THP-1 cells by Western blotting and the culture supernatant by enzyme-linked immunosorbent assay analysis were observed at 3 h after cocultivation. In addition, the increased expression of ASC and NLRP3, which make up an inflammasome complex, was also observed at 3 h after cocultivation. To confirm the caspase-1 activation and IL-1β production via the NLRP3 inflammasome in THP-1 cells triggered by N. fowleri trophozoites, THP-1 cells were pretreated with several inhibitors. The inhibition assay showed that CA-074 (a cathepsin B inhibitor), glybenclamide (an NLRP3 molecule inhibitor), and N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) reduced the levels of caspase-1 activation and IL-1β production from THP-1 cells. This study suggests that N. fowleri infection induces the NLRP3 inflammasome, which activates caspase-1 and subsequently produces IL-1β, thus resulting in inflammation.

Polyanhydride nanoparticles enhance activity of amoebastatic drugs against pathogenic Naegleria fowleri

Polyanhydride nanoparticles enhance activity of amoebastatic drugs against pathogenic Naegleria fowleri

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

Naegleria fowleri: nfa1 gene knock-down by double-stranded RNAs

Nfa1 protein expressed by the nfa1 gene that was cloned recently from pathogenic Naegleria fowleri was found in pseudopodia, especially food-cups, and concerned with a mechanism of pathogenicity of N. fowleri. In the present study, N. fowleri nfa1 gene was knocked down using double-stranded RNAs, and the expression of Nfa1 protein was observed. Using synthetic double-stranded RNA of the nfa1 gene in vitro, the nfa1 gene and Nfa1 protein were knocked down about 50.4 ± 3.1% and 52 ± 2%, respectively. These results suggest that RNA interference (RNAi) may be an effective technique for gene knock-down in N. fowleri trophozoites.

Molecular cloning and characterization of a cytosolic heat shock protein 70 from Naegleria fowleri

A gene encoding a cytosolic heat shock protein 70 from pathogenic Naegleria fowleri (Nf-cHSP70) was identified. The Nf-cHSP70 was 2,062 bp in length with an open reading frame of 1,980 bp encoding 659 amino acid residues. The deduced amino acid sequence of the gene shared high sequence identities with HSP70s from other parasitic organisms and mammals. The characteristic domains, including N-terminal ATPase domain, calmodulin-binding domain, and EE(D)VD motif, found in HSP70s were also well conserved in this gene. The recombinant Nf-cHSP70 protein showed strong antigenicity against the sera from mice experimentally infected with N. fowleri. Immunofluorescence assay showed that Nf-cHSP70 localized in cytosol of the parasite. The results from semi-quantitative RT-PCR and Western blot analyses demonstrated the expression levels of gene transcripts, and its products were significantly increased at high temperature (42 degrees C). The definitive biological roles of Nf-cHSP70 are not clear, but it may protect the parasite under environmental changes especially high temperature.

Morphologic and Molecular Identification of Naegleria dunnebackei n. sp. Isolated from a Water Sample

Naegleria dunnebackei n. sp., a new species of the free-living amoeboflagellate Naegleria, is described in this report. The organism was isolated from a water sample taken from drinking troughs associated with cases of primary amoebic meningoencephalitis in cattle at a ranch in southern California. The isolate grew at, but not above 37 degrees C, and did not kill young mice upon intranasal inoculation suggesting that it was not pathogenic. The new species combines morphological features of non-pathogenic Naegleria gruberi and pathogenic Naegleria fowleri. The trophic amoeba resembled other members of the genus, with a prominent vesicular nucleus and mitochondria with discoidal cristae; a Golgi apparatus was not observed by electron microscopy. The cyst stage had pores in the wall typical of those seen in pathogenic N. fowleri. Upon suspension in distilled water, amoebae transformed into temporary, non-feeding flagellates, mostly with two anterior flagella but occasionally with four. The rationale for its description as a new species was based upon sequencing of the 5.8S rDNA and internal transcribed spacers of the amoeba, which is similar to but not identical to that of Naegleria gallica, differing from that organism's DNA by six base pairs. Virus-like elements were found in the cytoplasm of trophic amoebae, often in association with crystalloids, and may be the cause of lysis of amoebae in culture.

PCR Detection and Analysis of the Free-Living Amoeba Naegleria in Hot Springs in Yellowstone and Grand Teton National Parks

Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.

Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri.

We previously cloned an antigenic gene (named nfa1) from a cDNA library of Naegleria fowleri by immunoscreening. The nfa1 gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of N. fowleri against Chinese hamster ovary (CHO) cells. Trophozoites of N. fowleri in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with N. fowleri, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. When examined using a light microscope, CHO cells grown in cocultures with N. fowleri trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with N. fowleri trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.

Effects of peracetic acid and monochloramine on the inactivation of Naegleria lovaniensis

Biocidal activities of monochloramine and peracetic acid were studied on cysts of Naegleria lovaniensis. Until recently the most commonly used biocide to disinfect cooling water systems was hypochlorite. Owing to its negative impact on the aquatic environment, ecologically less harmful alternatives have been sought. As the biocidal activity of monochloramine and peracetic acid makes them good candidates for inactivation of pathogenic Naegleria species, these biocides were tested against Naegleria lovaniensis, a relative of the pathogen Naegleria fowleri, as an alternative treatment to hypochlorite. Under laboratory conditions the biocidal activity of hypochlorite was 8- 10x stronger than that of the two investigated substances. Hypochlorite, at a concentration of 0.5 mg/L, killed 100% Naegleria lovaniensis after 1 h exposure (25 degrees C, pH 7.3- 7.4). To achieve similar results with monochloramine and peracetic acid, 3.94 mg/L or 5.33 mg/L had to be used respectively (25 degrees C, pH 8). It was known that the in situ biota of the biofilm, along with any organic material in the water column, had a negative impact on the efficiency of the biocides. There are, however, indications that the relative efficacy of monochloramine and peracetic acid was quite good under such conditions when compared with hypochlorite.

Free-living, amphizoic and opportunistic amebas.

Amebas belonging to the genera Naegleria, Acanthamoeba and Balamuthia are free-living, amphizoic and opportunistic protozoa that are ubiquitous in nature. These amebas are found in soil, water and air samples from all over the world. Human infection due to these amebas involving brain, skin, lung and eyes has increased significantly during the last 10 years. The epidemiology, immunology, protozoology, pathology, and clinical features of the infections produced by these protozoa differ strikingly. Infection by the pathogenic Naegleria fowleri is acquired by exposure to polluted water in ponds, swimming pools and man-made lakes. Raised temperatures during the hot summer months or warm water from power plants facilitate the growth of N. fowleri. N. fowleri is a thermophilic ameba that grows well in tropical and subtropical climates. The CNS infection, called Primary Amebic Meningoencephalitis (PAM), produced by N. fowleri is characterized by an acute fulminant meningoencephalitis leading to death 3-7 days after exposure. Victims are healthy, young individuals with a history of recent water-related sport activities. The portal of entry is the olfactory neuroepithelium. The pathologic changes are an acute hemorrhagic necrotizing meningoencephalitis with modest purulent exudate, mainly at the base of the brain, brain-stem and cerebellum. Trophozoites can be seen within the CNS lesions located mainly around blood vessels. Thus far 179 cases have been reported; 81 in the USA alone. Balamuthia mandrillaris and several species of Acanthamoeba are pathogenic "opportunistic" free-living amebas which cause Granulomatous Amebic Encephalitis (GAE) in humans and animals. GAE is an infection, usually seen in debilitated, malnourished individuals, in patients undergoing immunosuppressive therapy for organ transplants and in Acquired Immunodeficiency Syndrome (AIDS). The granulomatous component is negligible, particularly in immunocompromised individuals. Pathologically these amebas produce a patchy, chronic or subacute granulomatous encephalitis with the presence of trophozoites and cysts. The portal of entry is probably through the respiratory tract or an ulceration of the skin reaching the CNS by hematogenous spread. As of October 1, 1996, 166 cases (103 due to Acanthamoeba and 63 due to Balamuthia) of GAE have been reported from around the world. Of these 103 cases due to Acanthamoeba (72 have been reported in the USA alone, > 50 in AIDS). It is well known that several species of Acanthamoeba can also produce, chronic sight threatening ulceration of the cornea called Acanthamoeba keratitis (AK), mostly in contact lens wearers or in individuals with minor corneal abrasions. Hundreds of cases of AK have been documented world wide.

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.

Molecular Biology of DNA inAcanthamoeba, Amoeba, Entamoeba, and Naegleria

This chapter discusses the molecular biology of DNA in acanthameba, ameba, entameba, and naegleria. The chapter reviews a reasonably current review about gene structure and the overall organization of the DNA and its metabolism. The four genera of amebas that have been studied most extensively are described. The genus ameba includes large free-living organisms. The cell cycle is simple; replication is by binary fission and differentiation is unknown. These amebas are large enough to manipulate by various surgical procedures, and this attribute has been exploited extensively. Cultures typically are grown using the ciliated protozoan tetrahymena as the major food organism. The genus acanthameba includes small amebas that are ordinarily free-living, but can be opportunistic pathogens of animals and humans. Such organisms are considered amphizoic. In a few cases, infection has been lethal, but these organisms are ubiquitous in nature and most humans appear to have good resistance to infections. The taxonomy of acanthameba is confusing, and it is unclear whether pathogenicity is restricted to certain species or whether all strains are capable of it. acanthameba replicate by binary fission. They differentiate into dormant cysts (that is, they encyst) during adverse environmental conditions. The genus naegleria includes small amebas that can differentiate either into a cyst or a flagellated stage. In naegleria, it is clear that certain species are pathogenic and that other species are not. The pathogenic Naegleria fowleri is highly virulent and fatal to humans. The genus entameba is distinct from the rest because it includes anaerobes and parasites. The life cycle includes a phase of replication by binary fission and encystment, which occurs in response to environmental factors. Nuclear division can continue in the cyst in the absence of cytoplasmic division. Species are typically identified according to their hosts. Entameba histolytica, the human parasite, and Entameba invadens, a parasite of reptiles, are the two most commonly studied species. Both can be grown axenically under anaerobic conditions. Entameba moshkovskii is interesting because it appears to be free-living.

The association of naegleria fowleri with the chemical, microbiological and physical characteristics of South Australian water supplies

In this study, 102 water samples containing the pathogen Naegleria fowleri, were compared with control samples matched by sampling site and season. Generally, two controls were available for each sample where N. fowleri was detected. Univariate comparisons revealed a positive association of N. fowleri with: (a) water temperature; (b) maximum and minimum air temperature; (c) bacterial colony counts at 20 and 35°C; (d) the total coliform count; (e) amoebae other than Naegleria and (f) Naegleria sp. other than N. fowleri. By comparison, negative associations were evident with free and total chlorine residuals. A conditional logistic regression analysis indicated that N. fowleri isolation was positively and independently associated with: (a) water temperature; (b) maximum air temperature; (c) bacterial colony counts at 35°C; and (d) low free chlorine residual. Negative associations were found with high temperature tolerant Naegleria and Acanthamoeba, but it is considered that these findings may reflect laboratory technique, not genuine ecological relationships. Evidence is presented that the probability of detecting N. fowleri would vary markedly with the changes in means water temperature and maximum daily air temperature that occur by season. On the basis of temperature variations alone, and assuming a constant free chlorine residual, it is estimated that the probability of N. fowleri occurring (at detectable levels) would be 53 times higher in summer, eleven times higher in autumn, and six times higher in spring, than it would be in the winter. These are very approximate estimations which may not apply to an individual sampling site, nor to periods of extreme temperature. While this is a preliminary study, statistical associations of N. fowleri with a variety of ecological measures have been quantified. More comprehensive and detailed studies are required to further clarify the ecological relationships of this organism.

The Role of Free-Living Amoebae Occurring in Heated Effluents as Causative Agents of Human Disease

It has been shown that several pathogenic organisms may be frequently found in thermal effluents and cooling systems of coal fired power plants. One of them is pathogenic Naegleria fowleri, the causative agent of an acute fatal human disease - primary amoebic meningoencephalitis (PAM). In our study two out of eight power plants investigated, harbored pathogenic N. fowleri in heated water or cooling towers. The occurrence of this organism was related to elevated temperatures. No significant correlation was found for other biological and chemical parameters. In addition, pathogenic Acanthamoeba which causes granulomatous amoebic encephalitis (GAE) was found in the tested heated effluents from coal fired power plants. Non-pathogenic strains of N. fowleri as well as other free-living and “harmless” amoebae were also very abundant in effluents from all investigated coal fired power plants and cooling towers. It has been reported that several species of nonpathogenic amoebae were isolated from humidifiers and air conditioning systems. Serological testing of symptomatic human subjects has indicated that these organisms may be one of the causative agents of hypersensitivity pneumonitis. An experimental study performed in our laboratory involved testing of guinea pigs sensitized by injection of axenic, non-pathogenic N. gruberi. Delayed onset skin reactivity was apparent in all animals injected with the antigen. Antibodies were detected in all sensitized animals. Bronchial provocation challenge employed to investigate pulmonary hypersensitivity was also used, and yielded positive results. All the sensitized animals displayed delayed onset respiratory responses. The results of this study indicate that not only pathogenic but also non-pathogenic free-living amoebae may be important causative agents of human disease. The occurrence of these organisms in cooling systems from coal fired power plants indicates that these facilities may be an important source of infection.