Smoke on the Water: Comparative Assessment of Combined Thermal Shock Treatments for Control of Invasive Asian Clam, Corbicula fluminea.

Suppression of established populations of invasive alien species can be a complex and expensive process, which is frequently unsuccessful. The Asian clam, Corbicula fluminea (Müller, 1774), is considered a high impact invader that can adversely alter freshwater ecosystems and decrease their socioeconomic value. To date, C. fluminea continues to spread and persist within freshwater environments worldwide, despite repeated management attempts to prevent dispersal and suppress established populations. As extensive C. fluminea beds can often become exposed during low-water conditions, the direct application of hot or cold thermal shock treatments has been proposed as suitable mechanism for their control. Further, mechanical substrate disturbance may enhance the efficacy of thermal shock treatments by facilitating exposures to multiple layers of buried clams. In the present study, we advanced these methods by assessing combined applications of both hot and cold thermal shock treatments for control of C. fluminea, using steam spray (≥100 °C; 350 kPa), low- or high-intensity open-flame burns (~1000 °C) and dry ice (-78 °C). In a direct comparison of raking combined with hot thermal shock applications, both steam and high-intensity open-flame treatments tended to be most effective, especially following multiple applications. In addition, when hot thermal treatments are followed by a final cold shock (i.e. dry ice), steam treatments tended to be most effective. Further, when dry ice was applied either alone or prior to an application of a hot shock treatment, substantial if not complete C. fluminea mortality was observed. Overall, this study demonstrated that combined applications of hot and cold thermal shock treatments, applied following the disruption of the substrate, can substantially increase C. fluminea mortality compared to separate hot or cold treatments.

Using structured eradication feasibility assessment to prioritize the management of new and emerging invasive alien species in Europe.

Prioritizing the management of invasive alien species (IAS) is of global importance and within Europe integral to the EU IAS regulation. To prioritize management effectively, the risks posed by IAS need to be assessed, but so too does the feasibility of their management. While the risk of IAS to the EU has been assessed, the feasibility of management has not. We assessed the feasibility of eradicating 60 new (not yet established) and 35 emerging (established with limited distribution) species that pose a threat to the EU, as identified by horizon scanning. The assessment was carried out by 34 experts in invasion management from across Europe, applying the Non-Native Risk Management scheme to defined invasion scenarios and eradication strategies for each species, assessing the feasibility of eradication using seven key risk management criteria. Management priorities were identified by combining scores for risk (derived from horizon scanning) and feasibility of eradication. The results show eradication feasibility score and risk score were not correlated, indicating that risk management criteria evaluate different information than risk assessment. In all, 17 new species were identified as particularly high priorities for eradication should they establish in the future, whereas 14 emerging species were identified as priorities for eradication now. A number of species considered highest priority for eradication were terrestrial vertebrates, a group that has been the focus of a number of eradication attempts in Europe. However, eradication priorities also included a diverse range of other taxa (plants, invertebrates and fish) suggesting there is scope to broaden the taxonomic range of attempted eradication in Europe. We demonstrate that broad scale structured assessments of management feasibility can help prioritize IAS for management. Such frameworks are needed to support evidence-based decision-making.

Steam and Flame Applications as Novel Methods of Population Control for Invasive Asian Clam (Corbicula fluminea) and Zebra Mussel (Dreissena polymorpha).

Control strategies for established populations of invasive alien species can be costly and complex endeavours, which are frequently unsuccessful. Therefore, rapid-reaction techniques that are capable of maximising efficacy whilst minimising environmental damage are urgently required. The Asian clam (Corbicula fluminea Müller, 1774), and the zebra mussel (Dreissena polymorpha Pallas, 1771), are invaders capable of adversely affecting the functioning and biodiversity of freshwater ecosystems. Despite efforts to implement substantial population-control measures, both species continue to spread and persist within freshwater environments. As bivalve beds often become exposed during low-water conditions, this study examined the efficacy of steam-spray (≥100 °C, 350 kPa) and open-flame burn treatments (~1000 °C) to kill exposed individuals. Direct steam exposure lasting for 5 min caused 100% mortality of C. fluminea buried at a depth of 3 cm. Further, combined rake and thermal shock treatments, whereby the substrate is disturbed between each application of either a steam or open flame, caused 100% mortality of C. fluminea specimens residing within a 4-cm deep substrate patch, following three consecutive treatment applications. However, deeper 8-cm patches and water-saturated substrate reduced maximum bivalve species mortality rates to 77% and 70%, respectively. Finally, 100% of D. polymorpha specimens were killed following exposure to steam and open-flame treatments lasting for 30 s and 5 s, respectively. Overall, our results confirm the efficacy of thermal shock treatments as a potential tool for substantial control of low-water-exposed bivalves. Although promising, our results require validation through upscaling to field application, with consideration of other substrate types, increased substrate depth, greater bivalve densities, non-target and long-term treatment effects.

Using open-source software and digital imagery to efficiently and objectively quantify cover density of an invasive alien plant species.

The most commonly used method for measuring vegetation cover is visual estimation, which is highly subjective, potentially leading to measurement errors. This poses serious implications to the assessment and continued management of plant species cover, for example in the control of invasive plant species. Morphological analysis of digital imagery has, to date, been primarily applied in the classification of landscape features. Our novel application of morphological image analysis provides an objective method for detection and accurate cover assessment of an invasive alien plant species (IAS), giving reduced measurement errors when compared to visual estimation. Importantly, this method is entirely based on free software. Guidos Toolbox is a collection of generic raster image processing routines, including Morphological Spatial Pattern Analysis (MSPA), which classifies and quantifies features according to shape. MSPA was employed in this study to detect and quantify cover of invasive Petasites pyrenaicus (Winter heliotrope) in digital images of 1 m × 1 m plots. Its efficacy was compared to that of two other methods- GIS Digitisation (used as an accurate baseline) and Visual Estimation (standard method). We tested the limit of MSPA usability on images of varying complexity, i.e. "simple", intermediate" or "complex", depending on presence/absence of other vascular plant species and the species richness of plot. Our results show good agreement between all three methods. MSPA measurement of P. pyrenaicus cover was most closely aligned with the GIS Digitisation (concordance correlation coefficients of 0.966). Visual Estimation was less closely aligned with GIS Digitisation (concordance correlation coefficients of 0.888). However, image complexity resulted in differing levels of agreement; with the closest agreement being achieved between MSPA and GIS Digitisation when used on images of lower and higher complexity. MSPA consistently provides higher accuracy and precision for P. pyrenaicus cover measurement than the standard Visual Estimation method. Our methodology is applicable to a range of focal vegetation species, both herbaceous and graminoid. Future application of MSPA for larger-scale surveying and monitoring via remote sensing is discussed, potentially reducing resource demands and increasing cover measurement consistency and accuracy. We recommend this method forms part of vegetation management toolkits for not only environmental managers, but for anyone concerned with plant cover assessment, from agricultural systems to sustainable resource use.

The effectiveness of disinfectant and steam exposure treatments to prevent the spread of the highly invasive killer shrimp, Dikerogammarus villosus.

Biosecurity protocols designed to prevent the spread of invasive alien species (IAS) are now an essential aspect of IAS management. However, the effectiveness of various biosecurity treatments requires further exploration. Killer shrimp, Dikerogammarus villosus, a notoriously high impact and ecosystem destabilising invader, has rapidly spread across Europe, and is of concern to invade Northern America. In this study, we examine the effectiveness of three commonly used, broad-spectrum disinfectants to cause mortality of D. villosus: Virasure Aquatic, Virkon Aquatic, and Virkon S. Immersion and spray treatments of 1%, 2% and 4% disinfectant solutions were examined for applications of up to 300 secs immersion and for up to ten consecutive sprays. Furthermore, we assessed the effectiveness of steam (≥100 °C) treatments for up to 120 secs. For all disinfectants, immersion in 1% solutions caused 100% mortality at ≥120 secs. At higher concentrations, shorter immersion times caused complete mortality: 60 and 15 secs for 2% and 4% solutions, respectively. Five sprays of 2% and 4% solutions resulted in 100% mortality, for all disinfectants. Direct steam exposure was highly effective, with complete D. villosus mortality occurring at ≥10 secs. Overall, brief exposure to broad-spectrum disinfectants and direct steam could be used to limit D. villosus spread.

Temperature rise and parasitic infection interact to increase the impact of an invasive species.

Invasive species often detrimentally impact native biota, e.g. through predation, but predicting such impacts is difficult due to multiple and perhaps interacting abiotic and biotic context dependencies. Higher mean and peak temperatures, together with parasites, might influence the impact of predatory invasive host species additively, synergistically or antagonistically. Here, we apply the comparative functional response methodology (relationship between resource consumption rate and resource supply) in one experiment and conduct a second scaled-up mesocosm experiment to assess any differential predatory impacts of the freshwater invasive amphipod Gammarus pulex, when uninfected and infected with the acanthocephalan Echinorhynchus truttae, at three temperatures representative of current and future climate. Individual G. pulex showed Type II predatory functional responses. In both experiments, infection was associated with higher maximum feeding rates, which also increased with increasing temperatures. Additionally, infection interacted with higher temperatures to synergistically elevate functional responses and feeding rates. Parasitic infection also generally increased Q10 values. We thus suggest that the differential metabolic responses of the host and parasite to increasing temperatures drives the synergy between infection and temperature, elevating feeding rates and thus enhancing the ecological impact of the invader.

Comparison of diagnostic techniques for the detection of Cryptosporidium oocysts in animal samples.

While a large number of laboratory methods for the detection of Cryptosporidium oocysts in faecal samples are now available, their efficacy for identifying asymptomatic cases of cryptosporidiosis is poorly understood. This study was carried out to determine a reliable screening test for epidemiological studies in livestock. In addition, three molecular tests were compared to identify Cryptosporidium species responsible for the infection in cattle, sheep and horses. A variety of diagnostic tests including microscopic (Kinyoun's staining), immunological (Direct Fluorescence Antibody tests or DFAT), enzyme-linked immunosorbent assay (ELISA), and molecular methods (nested PCR) were compared to assess their ability to detect Cryptosporidium in cattle, horse and sheep faecal samples. The results indicate that the sensitivity and specificity of each test is highly dependent on the input samples; while Kinyoun's and DFAT proved to be reliable screening tools for cattle samples, DFAT and PCR analysis (targeted at the 18S rRNA gene fragment) were more sensitive for screening sheep and horse samples. Finally different PCR primer sets targetedat the same region resulted in the preferential amplification of certain Cryptosporidium species when multiple species were present in the sample. Therefore, for identification of Cryptosporidium spp. in the event of asymptomatic cryptosporidiosis, the combination of different 18S rRNA nested PCR primer sets is recommended for further epidemiological applications and also tracking the sources of infection.

Ecotoxicological impact of Zequanox®, a novel biocide, on selected non-target Irish aquatic species.

Effective, species-specific zebra mussel control is needed urgently for Ireland׳s freshwater bodies, which became infested with non-native zebra mussels in the 1990s. Zequanox®, a newly commercialized product for zebra and quagga mussel control, is composed of dead Pseudomonas fluorescens CL 145A cells. This paper describes ecotoxicology tests on three representative native Irish freshwater species: Anodonta (duck mussel), Chironomus plumosus (non-biting midge), and Austropotamobius pallipes (white-clawed crayfish). The species were exposed to Zequanox in a 72-h static renewal toxicity test at concentrations of 100-750mg active ingredient per liter (mga.i./L). Water quality parameters were measured every 12-24h before and after water and product renewal. After 72h, endpoints were reported as LC10, LC50, and LC100. The LC50 values derived were (1) Anodonta: ≥500mga.i./L (2) C. plumosus: 1075mga.i./L, and (3) A. pallipes: ≥750mga.i./L. These results demonstrate that Zequanox does not negatively affect these organisms at the concentration required for >80percent zebra mussel mortality (150mg a.i/L) and the maximum allowable treatment concentration in the United Sates (200mga.i./L). They also show the overall species-specificity of Zequanox, and support its use in commercial facilities and open waters.

Municipal wastewater treatment plants as pathogen removal systems and as a contamination source of noroviruses and Enterococcus faecalis.

Municipal wastewater treatment plants play a crucial role in reducing the microbial and pathogen load of human wastes before the end-products are discharged to surface waters (final effluent) or land spread (biosolids). This study investigated the occurrence frequency of noroviruses, Enterococcus faecalis and Enterococcus faecium in influent, final effluent and biosolids from four secondary wastewater treatment plants in northwestern Ireland (plants A-D) and observed the seasonal and spatial variation of the plant treatment efficiencies in the pathogen removals. It was noted that norovirus genogroup II was more resistant to the treatment processes than the norovirus genogroup I and other active viral particles, especially those in the discharge effluents. The percolating biofilm system at plant D resulted in better effluent quality than in the extended aerated activated sludge systems (plants A and B); primary biosolids produced at plant D may pose a higher health risk to the locals. The spread of norovirus genogroup II into the environment, irrespective of the wastewater treatment process, coincides with its national clinical predominance over norovirus genogroup I. This study provides important evidence that municipal wastewater treatment plants not only achieve pathogen removal but can also be the source of environmental pathogen contamination.

Municipal wastewater treatment plants as removal systems and environmental sources of human-virulent microsporidian spores.

Municipal wastewater treatment plants play a vital role in reducing the microbial load of sewage before the end-products are discharged to surface waters (final effluent) or local environments (biosolids). This study was to investigate the presence of human-virulent microsporidian spores (Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon hellem) and enterococci during treatment processes at four Irish municipal secondary wastewater treatment plants (plants A-D). Microsporidian abundance was significantly related to seasonal increase in water temperature. Plant A had the least efficient removal of E. intestinalis spores (32%) in wastewater, with almost 100% removal at other plants both in April and July. Some negative removal efficiencies were obtained for E. bieneusi (at plants C and D, -100%) and for E. hellem (at plants A and D, -90% and -50%). In addition, a positive correlation was found between the levels of enterococci and E. bieneusi in July (r (s) = 0.72, P < 0.05). In terms of the dewatered biosolids, a median concentration as high as 32,000 spores/Kg of E. hellem was observed at plant D in July. Plant C sewage sludge contained the lowest microsporidian loadings (E. bieneusi; 450 spores/L and 1,000 spores/L in April and July, respectively). This study highlights the seasonal variation in concentrations of microsporidian spores in the incoming sewage. Spores in final effluents and dewatered biosolids can be the source of human-virulent microsporidian contamination to the local environment. This emphasizes a considerably high public health risk when sewage-derived biosolids are spread during summer months. This study also suggested enterococci as a potential indicator of the presence of microsporidian spores in wastewater, especially for E. bieneusi.

Relationships among bather density, levels of human waterborne pathogens, and fecal coliform counts in marine recreational beach water.

During summer months, samples of marine beach water were tested weekly for human waterborne pathogens in association with high and low bather numbers during weekends and weekdays, respectively. The numbers of bathers on weekends were significantly higher than on weekdays (P < 0.001), and this was associated with a significant (P < 0.04) increase in water turbidity. The proportion of water samples containing Cryptosporidium parvum, Giardia duodenalis, and Enterocytozoon bieneusi was significantly higher (P < 0.03) on weekends than on weekdays, and significantly (P < 0.01) correlated with enterococci counts. The concentration of all three waterborne pathogens was significantly correlated with bather density (P < 0.01). The study demonstrated that: (a) human pathogens were present in beach water on days deemed acceptable for bathing according to fecal bacterial standards; (b) enterococci count was a good indicator for the presence of Cryptosporidium, Giardia, and microsporidian spores in recreational marine beach water; (c) water should be tested for enterococci during times when bather numbers are high; (d) re-suspension of bottom sediments by bathers caused elevated levels of enterococci and waterborne parasites, thus bathers themselves can create a non-point source for water contamination; and (e) exposure to recreational bathing waters can play a role in epidemiology of microsporidiosis. In order to protect public health, it is recommended to: (a) prevent diapered children from entering beach water; (b) introduce bather number limits to recreational areas; (c) advise people with gastroenteritis to avoid bathing; and (d) use showers prior to and after bathing.

Propagation of human enteropathogens in constructed horizontal wetlands used for tertiary wastewater treatment.

Constructed subsurface flow (SSF) and free-surface flow (FSF) wetlands are being increasingly implemented worldwide into wastewater treatments in response to the growing need for microbiologically safe reclaimed waters, which is driven by an exponential increase in the human population and limited water resources. Wastewater samples from four SSF and FSF wetlands in northwestern Ireland were tested qualitatively and quantitatively for Cryptosporidium spp., Giardia duodenalis, and human-pathogenic microsporidia, with assessment of their viability. Overall, seven species of human enteropathogens were detected in wetland influents, vegetated areas, and effluents: Cryptosporidium parvum, C. hominis, C. meleagridis, C. muris, G. duodenalis, Encephalitozoon hellem, and Enterocytozoon bieneusi. SSF wetland had the highest pathogen removal rate (i.e., Cryptosporidium, 97.4%; G. duodenalis, 95.4%); however, most of these values for FSF were in the negative area (mean, -84.0%), meaning that more pathogens were discharged by FSF wetlands than were delivered to wetlands with incoming wastewater. We demonstrate here that (i) the composition of human enteropathogens in wastewater entering and leaving SSF and FSF wetlands is highly complex and dynamic, (ii) the removal and inactivation of human-pathogenic microorganisms were significantly higher at the SSF wetland, (iii) FSF wetlands may not always provide sufficient remediation for human enteropathogens, (iv) wildlife can contribute a substantial load of human zoonotic pathogens to wetlands, (v) most of the pathogens discharged by wetlands were viable, (vi) large volumes of wetland effluents can contribute to contamination of surface waters used for recreation and drinking water abstraction and therefore represent a serious public health threat, and (vii) even with the best pathogen removal rates achieved by SSF wetland, the reduction of pathogens was not enough for a safety reuse of the reclaimed water. To our knowledge, this is the first report of C. meleagridis from Ireland.

Fate of Cryptosporidium parvum and Cryptosporidium hominis oocysts and Giardia duodenalis cysts during secondary wastewater treatments.

This study investigates the fate of Cryptosporidium parvum and C. hominis oocysts and Giardia duodenalis cysts at four Irish municipal wastewater treatment plants (i.e., Plant A, B, C, and D) that utilize sludge activation or biofilm-coated percolating filter systems for secondary wastewater treatment. The fate of these pathogens through the sewage treatment processes was determined based on their viable transmissive stages, i.e., oocysts for Cryptosporidium and cysts for Giardia. Analysis of final effluent indicated that over 97% of viable oocysts and cysts were eliminated, except at Plant C, which achieved only 64% of oocyst removal. A significant correlation between the removal of oocysts and cysts was found at Plants A, B, and D (R = 0.98, P < 0.05). All sewage sludge samples were positive for C. parvum and C. hominis, and G. duodenalis, with maximum concentrations of 20 oocysts and eight cysts per gram in primary sludge indicating the need for further sludge sanitization treatments. This study provides evidence that C. parvum and C. hominis oocysts and G. duodenalis cysts are present throughout the wastewater processes and in end-products, and can enter the aquatic environment with consequent negative implications for public health.

Human zoonotic enteropathogens in a constructed free-surface flow wetland.

Effluents from a small-scale free-surface flow constructed wetland, used for polishing of secondary treated wastewater, contained significantly higher concentrations of potentially viable Giardia duodenalis cysts and Enterocytozoon bieneusi spores than did wetland influents consisting of secondary treated wastewater. Zoonotic Assemblage A of G. duodenalis cysts was identified in wetland inflows, while Assemblage A and two nonhuman infective Assemblages (i.e., C, and E) were present in wetland effluents. E. bieneusi spores represented genotype K based on DNA sequencing analysis of internal transcribed spacer. The study demonstrated that: (1) free-surface flow small-scale constructed wetlands may not provide sufficient remediation for human zoonotic protozoa and fungi present in secondary treated wastewater; (2) dogs and livestock can substantially contribute human-pathogenic protozoan and fungal microorganisms to engineered vegetated wetland systems; and (3) large volumes of wetland effluents can contribute to contamination of surface waters used for recreation and drinking water abstraction and therefore represent a serious public health threat.

Biomonitoring of surface and coastal water for Cryptosporidium, Giardia, and human-virulent microsporidia using molluscan shellfish.

Surface inland and coastal waters in Ireland were surveyed for the human waterborne enteropathogens; Cryptosporidium parvum, Giardia lamblia, Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi by utilizing bivalve mussel species, i.e., Mytilus edulis (blue mussel), Anodonta anatina (duck 'mussel', actually a unionid clam), and the invasive Dreissena polymorpha (zebra mussel) as biomonitors at twelve sites located in three Irish river-basin districts with various water-quality pressures. Biomolecular techniques were utilized to assess the presence and concentration of these pathogens. At least one pathogen species was detected in shellfish at each site. Cryptosporidium, implicated in several recent Irish gastrointestinal epidemics, was recorded at all sites subjected to agricultural runoff and at one sewage discharge site, linking source-track directly to human and animal fecal wastes. G. lamblia was present at eleven of the twelve sites in a range of concentrations. A coastal bay with raw urban sewage discharge was 100% positive for all analyzed enteropathogens. Overall, the results demonstrate long-term human enteropathogen contamination of Irish waters with consequent public-health risk factors for drinking-water abstraction and water-based activities.

Occurrence of Cryptosporidium and Giardia in sewage sludge and solid waste landfill leachate and quantitative comparative analysis of sanitization treatments on pathogen inactivation.

Circulation of Cryptosporidum and Giardia in the environment can be facilitated by spreading of sewage sludge on agricultural or livestock grazing lands or depositing in landfills. Solid waste landfill leachate and sewage sludge samples were quantitatively tested for C. parvum and C. hominis oocysts, and G. lamblia cysts by the combined multiplexed fluorescence in situ hybridization (FISH) and immunofluorescent antibody (IFA) method. Subsequently, the effects of four sanitization treatments (i.e., ultrasound and microwave energy disintegrations, and quicklime and top-soil stabilization) on inactivation of these pathogens were determined. The landfill leachate samples were positive for Giardia, and sewage sludge samples for both Cryptosporididium and Giardia. The overall concentration of G. lamblia cysts (mean; 24.2/g) was significantly higher (P<0.01) than the concentration of C. parvum and C. hominis oocysts (mean; 14.0/g). Sonication reduced the load of G. lamblia cysts to non-detectable levels in 12 of 21 samples (57.1%), and in 5 of 6 samples (83.3%) for C. parvum and C. hominis. Quicklime stabilization treatment was 100% effective in inactivation of Cryptosporidium and Giardia, and microwave energy disintegration lacked the efficacy. Top-soil stabilization treatment reduced gradually the load of both pathogens which was consistent with the serial dilution of sewage sludge with the soil substrate. This study demonstrated that sewage sludge and landfill leachate contained high numbers of potentially viable, human-virulent species of Cryptosporidium and Giardia, and that sonication and quicklime stabilization were the most effective treatments for sanitization of sewage sludge and solid waste landfill leachates.

Bather density and levels of Cryptosporidium, Giardia, and pathogenic microsporidian spores in recreational bathing water.

The study demonstrated that the resuspension of bottom sediments caused by bathers and their direct microbial input resulted in elevated levels of Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores, particularly Enterocytozoon bieneusi, in recreational beach water on days deemed acceptable for bathing by fecal bacterial standards.

Quantitative evaluation of the impact of bather density on levels of human-virulent microsporidian spores in recreational water.

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.

Quality of reclaimed waters: a public health need for source tracking of wastewater-derived protozoan enteropathogens in engineered wetlands.

Demand for high-quality drinking and recreational water rises exponentially owing to global demographic growth in the human population, reinforcing an urgent need for microbiologically safe reclaimed water. However, constructed wetlands, implemented into municipal wastewater treatment, may not provide substantial remediation for human protozoan enteropathogens such as Cryptosporidium, Giardia and human-virulent microsporidia. Improving reclaimed water quality by lowering faecal coliforms is not a sound solution for these pathogens. Current advances in molecular technology can benefit public health in developing and developed countries by changing the conceptual research framework for wastewater-receiving wetlands from 'pathogen removal' to 'pathogen source tracking' efforts.

Human-virulent microsporidian spores in solid waste landfill leachate and sewage sludge, and effects of sanitization treatments on their inactivation.

Solid waste landfill leachate and sewage sludge samples were quantitatively tested for viable Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi spores by the multiplexed fluorescence in situ hybridization (FISH) assay. The landfill leachate samples tested positive for E. bieneusi and the sludge samples for E. bieneusi and E. intestinalis. The effects of four sanitization treatments on the inactivation of these pathogens were assessed. Depending on the variations utilized in the ultrasound disintegration, sonication reduced the load of human-virulent microsporidian spores to nondetectable levels in 19 out of 27 samples (70.4%). Quicklime stabilization was 100% effective, whereas microwave energy disintegration was 100% ineffective against the spores of E. bieneusi and E. intestinalis. Top-soil stabilization treatment gradually reduced the load of both pathogens, consistent with the serial dilution of sewage sludge with the soil substrate. This study demonstrated that sewage sludge and landfill leachate contained high numbers of viable, human-virulent microsporidian spores, and that sonication and quicklime stabilization were the most effective treatments for the sanitization of sewage sludge and solid waste landfill leachates. Multiplexed FISH assay is a reliable quantitative molecular fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores in environmental samples.