Thesis (M.Sc.) from the Department of Zoology, University of Adelaide, 1975.

The free-living amoeba and causative agent of Primary Amoebic Meningoencephalitis, Naegleria fowleri, has three life stages: the trophozoite, the flagellate, and the cyst. This study examined the ability of the cyst to attach to, excyst upon, and destroy cell cultures grown to confluent monolayers, and to cause Primary Amoebic Meningoencephalitis in a murine animal model. The co-culture of cysts with P388D.1, CHME3, Vero, human nasal epithelial, and rat primary mixed glial cells resulted in destruction of the monolayer of all cell types once the cysts attached and excysted. One day post exposure to cysts, the mixed glial cells exhibited a two-fold increase in lactate dehydrogenase (LDH) release compared to cells without cysts, and on day eight post exposure, showed a nearly four-fold increase in LDH. In this study, the cysts of N. fowleri were shown not to be infective in vivo in a murine model using B6C3F1 male mice. The mediation of the encystment process by the intracellular concentration of the secondary messenger cAMP, as described in other closely related genera and species of amoeba, was also investigated. Encystment of N. fowleri was shown to be mediated at least in part by the secondary messenger cAMP by treating cultures of the trophozoite with 100 uM dipyridamole, an inhibitor of cAMP-specific phosphodiesterases. Dipyridamole (100 μM) increased the rate of encystment by nearly two-fold compared to 0.1% DMSO by the end of a five day period of observation. This suggests that cAMP is an essential mediator of the encystment process within Naegleria fowleri.

Emerging pathogens are organisms whose incidence has increased within the past two decades. In the last 40 years, several pathogens have emerged to cause infectious waterborne and foodborne diseases, thus causing a significant public health concern. Enterobacter sakazakii and Naegleria fowleri are emerging pathogens that have been documented to cause fatal infections. E. sakazakii is an emerging foodborne pathogen that represents a significant health risk by causing infections resulting in septicemia, meningitis and necrotizing enterocolitis in neonates, premature infants and also elderly immunocompromised individuals. Naegleria fowleri is a water-based protozoan flagellate that is the cause of primary amoebic meningoencephalitis; a fatal disease that mostly infects children and young adults through water-related recreational activities. The focus of this dissertation is to identify environmental reservoirs of Enterobacter sakazakii and to determine inactivation strategies to control Naegleria fowleri by chlorine and ultraviolet disinfection. In Appendix A, samples from various household kitchens were collected to determine the presence of E.sakazakii. The highest percentage of E.sakazakii was isolated from kitchen sponges (8%; n=50) and dishrags (10%; n=50). This study provided information on the presence of E.sakazakii on environmental surfaces in the kitchen. In Appendix B, our recent research has determined that N. fowleri is present in 8% (n=143) of municipal drinking water wells in central and southern Arizona. Therefore, guidelines need to be established for treatment of water with various disinfectants to control the growth and proliferation of N.fowleri. In Appendix C, the Ct values (concentration (mg/l) × exposure time) for chlorine inactivation of N. fowleri trophozoites and cysts were determined using the Efficiency Hom Kinetic Model (EHM). The Ct values for 99% inactivation of trophozoites and cysts were estimated to be 9 and 31, respectively. The ultraviolet light dose required for the 99% inactivation of N.fowleri trophozoites and cysts was determined to be 63 mW.sec/cm² and 13 mW.sec/cm², respectively.

No description available.

Dans l'objectif d'anticiper et de réduire la prolifération de l'amibe pathogène Naegleria fowleri dans les circuits de refroidissement de certaines centrales électriques, notre travail vise à mieux comprendre l'écologie de cette amibe dans des environnements complexes tels que les biofilms d'eau douce récemment reconnus comme niche écologique préférentielle des amibes libres. Des essais de laboratoire ont été réalisés pour déterminer l'impact des facteurs environnementaux naturels et anthropiques: température, nature du matériau support de la formation du biofilm, charge nutritionnelle et monochloramination sur le comportement et le devenir de Naegleria fowleri dans le biofilm. Ces travaux ont permis de démontrer que la survie, l'implantation, la croissance, le maintien et le déclin de Naegleria fowleri dans les biofilms sont principalement gouvernés par la concomitance des facteurs température et ressource nutritive. Les autres facteurs: nature du matériau, désinfection à la monochloramine et compétition amibienne, se présentent plutôt comme des paramètres de perturbation ou d'inhibition de cette dynamique. Par ailleurs, les résultats obtenus sur la colonisation du biofilm par les amibes confortent le rôle prépondérant de cet habitat comme réservoir naturel des amibes libres et Naegleria fowleri / This study is aiming at preventing and reducing the proliferation of the pathogenic free-living amoeba Naegleria fowleri in several power plant cooling circuits. This work contributes to provide a better understanding of the ecology of this amoeba in complex environments such as freshwater biofilms, which recently has been recognized as privileged ecological niche for free-living amoebae. Laboratory tests were conducted to determine the impact of environmental factors such as temperature, type of support material for the biofilm formation, nutritional resources and monochloramination treatment on the behavior and the fate of Naegleria fowleri in the biofilm. This work has demonstrated that the survival, implantation, maintain, growth and decline of Naegleria fowleri in biofilms are mainly governed by a combination of the temperature and nutritional resource factors. The other factors: type of support material, monochloramination treatment, and amoebic competition, appeared rather as disruptive or inhibitory parameters of this dynamic. Moreover, the obtained results for the amoebic colonization of the biofilm matrix confirm the crucial role of this habitat as natural reservoir for free-living amoebae and Naegleria fowleri

The amoebae Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are free-living amoebae found in both water and soil. They are opportunistic pathogens in humans. Acanthamoeba is the most common cause of illness, usually infecting the eyes and sometimes causing a sight-threatening keratitis. Acanthamoeba spp. and B. mandrillaris can cause granulomatous amoebic encephalitis, in addition to infections of the lungs and skin. N. fowleri causes primary amoebic meningoencephalitis . There is little known regarding the ecology and occurrence of these organisms. A total of 36 high-use recreational surface waters in Arizona were surveyed over a period of two years to assess the occurrence of N. fowleri and seasonal and environmental factors. Overall, 9.3% of the warm weather samples collected were positive for N. fowleri, whereas 16.3% of the samples were positive during cold weather. Although the presence of N. fowleri could not be significantly correlated with physical and chemical parameters such as temperature, pH, turbidity, conductivity, and the presence of heterotrophic bacteria, total coliforms, and Escherichia coli, a weak correlation (0.52) with live amoebic activity was observed. Five lakes to the north and northeast of Phoenix tested positive for the N. fowleri on more than one occasion over multiple seasons. Finished drinking water samples (n= 785) from a municipal potable distribution system were evaluated for the presence of N. fowleri, B. mandrillaris and Acanthamoeba spp. from 18 different regions during three different sampling periods. Physical and chemical parameters were also evaluated but provided no significant correlations with the occurrence of amoebae or indicator organisms. A total of 138 samples (17.9%) were positive for viable amoebae in distribution water with more than an adequate chlorine residual (average of 0.86 mg/L). Microorganisms that are typically used to monitor microbial water quality such as coliforms and E. coli would likely not be found under these circumstances. Clusters with three or more samples testing positive for viable amoebae per region were observed during all three periods. Viable amoebae may not only provide a better assessment of the microbial quality of water, but such clustering could reveal areas with potential water quality issues within the distribution system.

abstract: The deterioration of drinking-water quality within distribution systems is a serious cause for concern. Extensive water-quality deterioration often results in violations against regulatory standards and has been linked to water-borne disease outbreaks. The causes for the deterioration of drinking water quality inside distribution systems are not yet fully understood. Mathematical models are often used to analyze how different biological, chemical, and physical phenomena interact and cause water quality deterioration inside distribution systems. In this dissertation research I developed a mathematical model, the Expanded Comprehensive Disinfection and Water Quality (CDWQ-E) model, to track water quality changes in chloraminated water. I then applied CDWQ-E to forecast water quality deterioration trends and the ability of Naegleria fowleri (N.fowleri), a protozoan pathogen, to thrive within drinking-water distribution systems. When used to assess the efficacy of substrate limitation versus disinfection in controlling bacterial growth, CDWQ-E demonstrated that bacterial growth is more effectively controlled by lowering substrate loading into distribution systems than by adding residual disinfectants. High substrate concentrations supported extensive bacterial growth even in the presence of high levels of chloramine. Model results also showed that chloramine decay and oxidation of organic matter increase the pool of available ammonia, and thus have potential to advance nitrification within distribution systems. Without exception, trends predicted by CDWQ-E matched trends observed from experimental studies. When CDWQ-E was used to evaluate the ability N. fowleri to survive in finished drinking water, the model predicted that N. fowleri can survive for extended periods of time in distribution systems. Model results also showed that N. fowleri growth depends on the availability of high bacterial densities in the 105 CFU/mL range. Since HPC levels this high are rarely reported in bulk water, it is clear that in distribution systems biofilms are the prime reservoirs N. fowleri because of their high bacterial densities. Controlled laboratory experiments also showed that drinking water can be a source of N. fowleri, and the main reservoir appeared to be biofilms dominated by bacteria. When introduced to pipe-loops N. fowleri successfully attached to biofilms and survived for 5 months. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2010

Opportunistic pathogens (OPs) are of special concern for immunocompromised populations and are known to grow in both drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems, with aerosol inhalation and other non-ingestion exposures that are not addressed by existing regulatory frameworks. Factors enabling the growth of OPs in water distribution and premise (i.e., building) plumbing systems distributing reclaimed and other water sources systems are poorly understood especially for the emerging OP, Naegleria fowleri (i.e. brain-eating amoeba). Three phases of investigation were carried out to identify factors that facilitate the growth of OPs in main distribution and premise plumbing systems, with particular attention on reclaimed water systems, aging water mains, and private well systems. Phase one examined the role of biological treatment to remove organic carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed water distribution systems were employed to systematically examine the effects of prior granular activated carbon (GAC) biofiltration of the water; chlorine, chloramines, or no disinfectant, and water ages ranging up to 5 days. The second and third phases of research explored the role of nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal drinking water from a city grappling with aging water infrastructure and untreated private well water. Results from the simulated reclaimed water distribution systems suggested that biologically-active GAC filtration may unintentionally select for specific OPs, contrary to expectations and experiences with oligotrophic conditions in potable water systems. While GAC biofiltration was associated with lower total bacteria and Legionella spp. gene markers, there were no apparent benefits in terms of other OPs analyzed. Similarly, disinfectant treatments successful for controlling OPs in potable water were either ineffective or associated with increased levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water examined. In the potable water study, it was possible to recreate conditions associated with growth of N. fowleri in the aged main distribution system from where the water for the experiment was collected; including corroding iron mains, nitrification, and disinfectant decay. While the effects of nitrification could not be confirmed, there was a clear association of iron corrosion with N. fowleri proliferation. The role of iron was explored further in what, to the author's knowledge, was the first study of N. fowleri in private wells. Analysis of 40 wells found correlations between N. fowleri and stagnant iron levels, further supporting the hypothesis that iron corrosion or iron encourages the growth of N. fowleri, and, because wells are not routinely disinfected, not necessarily as a result of promoting disinfectant decay. As this study took place following a major flooding event, it provided insight not only into how surface water contamination may influence private well water microbial communities, but also added to the understanding that current recommendations for disinfecting private wells are inadequate and standards should be implemented to aid homeowners in the event of flooding. This exploratory research illuminated several factors influencing the OP growth in a range of water systems. Identifying key variables that control growth is crucial to improving the safety of these systems. / MS / Water borne bacteria that effect the immune systems of the sick, known as opportunistic pathogens (OPs), have become a major heath concern. These organisms are known to grow in drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems yet there are no regulations aimed at prevention. There is also limited knowledge on how premise plumbing and water sources effect the growth, population, and risk of infection by OPs, especially for Naegleria fowleri (i.e. brain-eating amoeba). An investigation was carried out in three parts to determine what influences the growth of OPs in water distribution and household plumbing systems, with particular attention to reclaimed water, municipal drinking water, and private well systems. Phase one examined the role of biological treatment to remove organic carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed water distribution systems were used to examine the effects of granular activated carbon (GAC) biofiltration of the water, disinfectants (chlorine, chloramines, or no disinfectant), and water ages ranging zero to five days. The second and third phases of research explored the role of nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal drinking water from a city with aging water infrastructure and untreated private well water. Results from the simulated reclaimed water distribution systems suggested that biologically-active GAC filtration may allow for the growth of specific OPs. While GAC biofiltration was associated with lower total bacteria and Legionella spp., there were no apparent benefits in reducing the presence of other OPs. Similarly, common disinfectant treatments for preventing OPs in drinking water were either ineffective or increased viii levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water. In the drinking water study, conditions were introduced to grow N. fowleri in aged drinking water distribution systems with the additions of corroding iron, nitrification (using nitrifying bacteria), and disinfectant. While the effects of nitrification could not be confirmed, there was a clear relationship between iron corrosion and N. fowleri growth. The role of iron was explored further in what, to the author’s knowledge, was the first study of N. fowleri in private wells. Forty wells were examined and the relationships between N. fowleri and stagnant iron levels supported the hypothesis that iron corrosion or iron increases the growth of N. fowleri. As this study took place following a major flooding event, it provided data not only into how surface water contamination may influence private well water microbial communities, but also added to the understanding that current recommendations for disinfecting private wells are inadequate and standards should be implemented to aid homeowners in the event of flooding. This exploratory research highlighted several variables that may allow for the growth of OPs in a range of water systems. Identifying key variables that control growth is crucial to improving the safety of these systems.

In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and Vero CCL-81, Chlorocebus sp. kidney epithelial cells, which were utilized as a positive control for Golgi expression. N. fowleri and N. lovaniensis whole-cell lysates had indications of a 110 kDa reduced protein, associated with the predicted molecular weights of the beta-COPI subunit of the COPI cis-Golgi vesicular transport complex with further Western immunoblot indication of a weak band around 25 kDa corresponding to rabbit polyclonal antibodies specific for ARF1. Serial Dilutions of Wheat Germ Agglutinin Alexa Fluor 488TM were performed on Vero cells, Naegleria fowleri 30894, and N. gruberi 30540 with 1:100 dilution of recommended stock dilution of WGA 488 determined for utilization in sequential immunofluorescence. Sequential immunofluorescence with Wheat Germ Agglutinin Alexa Fluor 488TM and then blocked with 3% BSA:PBS [wt/vol] dilution with subsequent incubation in rabbit anti-beta-COPI primary 1:250, and 1:1000 of Alexa Fluor 594 goat anti-rabbit secondary antibody exposure showed strong indications of organized cis- and trans-punctate Golgi body markers in close association in individual and dividing cells of Naegleria fowleri and conserved Golgi expression in the positive control Vero cells, but further experiments are necessary to verify this finding with N. fowleri.