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Microbiological & Chemical Exposure Assessment Research Division Publications: 2007

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This page lists publication titles, citations and abstracts produced by NERL's Microbiological & Chemical Exposure Assessment Research Division for the year 2007, organized by Publication Type. Your search has returned 199 Matching Entries.

See also Microbiological & Chemical Exposure Assessment Research Division citations with abstracts: 1999,  2000,  2001,  2002,  2003,  2004,  2005,  2006,  2007,  2008,  2009

Technical Information Manager: Angela Moore - (513) 569-7341 or moore.angela@epa.gov

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Presented/Published
BOOK CHAPTER Fecal Pollution of Water. 08/29/2008
SANTO-DOMINGO, J. W. AND N. ASHBOLT. Fecal Pollution of Water. Chapter N/A, Cutler J. Cleveland (ed.), Encyclopedia of Earth. National Council for Science and the Environment, Washington, D.C., DC, 1-12, (2008).
Abstract: Fecal pollution of water from a health point of view is the contamination of water with disease-causing organisms (pathogens) that may inhabit the gastrointestinal tract of mammals, but with particular attention to human fecal sources as the most relevant source of human illnesses globally. Ingestion of water contaminated with feces is responsible for a variety of diseases important to humans via what is known as the fecal-oral route of transmission. Food, air, soil, and all types of surfaces can also be important in the transmission of fecal pathogens, and thereby implicated in disease outbreaks. Most fecal microorganisms, however, are not pathogenic. Indeed, some are considered beneficial to the host as they can outcompete pathogens for space and nutrients, complement the biochemical potential of the host’s gastrointestinal tract, and help in the development of the host immune system. Nonetheless, animal feces can also carry a number of important frank and opportunistic pathogens, capable of inflicting debilitating illnesses and, in some cases, death. In 1998, it was estimated that 2.2 million deaths were associated with diarrhea each year, a good percentage of them due to fecal pollution of water, with the vast majority of victims being children in poor countries (WHO, 2000). This should not be a surprise as it has been estimated that more than 1 billion people worldwide lack access to safe drinking water, and more than 2 billion lack sanitation. Sadly, very little progress has been made in the last 20 years to ameliorate these problems, particularly due to the rapidly increasing global population. On the contrary, problems associated with fecal pollution of water are likely to worsen in coming decades, as more people are moving to coastal areas, most people now live in urban centers, many of which have out of control growth rates, and demands for animal meat products are increasing due to current trends in dietary regimes. Considering that per capita water availability and quantity are diminishing worldwide, it is reasonable to assume that fecal pollution of water is one of the most important and difficult challenges for future generations.

BOOK CHAPTER The Empact Beaches: A Case Study in Recreational Water Sampling 11/01/2007
WYMER, L. J. The Empact Beaches: A Case Study in Recreational Water Sampling. , Chapter 7, Larry Wymer (ed.), Statistical Framework for Recreational Water Quality Criteria and Monitoring. John Wiley and Sons, LTD, , Uk, 113-134, (2007).
Abstract: Various chapters describe sample and experimental design, use of a geometric mean or an arithmetic mean, modeling and forecasting, and risk assessment in relation to monitoring recreational waters for fecal indicators. All of these aspects of monitoring are dependent on the spatial and temporal distribution of fecal indicator bacteria in the water. Knowledge of the distribution of indicator bacteria in water is particularly important in sampling design for monitoring. The U.S. Environmental Protection Agency (EPA) conducted intensive water sampling studies during the summer of 2000 at five beaches in order to characterize temporal and spatial distribution of fecal indicator bacteria within the bathing areas of these beaches (Wymer et al., 2005, hereafter referred to as the EMPACT report). Study beaches encompassed a variety of environments (Table 1). During the months of July and August in 2000, water samples were collected at least twice daily from each of nine "fixed" locations in the water, as determined by a transect and zone (Figure 1). A transect consisted of an imaginary line through a fixed point on the beach perpendicular to the shoreline. A zone (or "depth zone") was defined as a contour line of equal water depth. As illustrated in Figure 1, each sampling location was defined by the intersection of transect and zone on a grid comprising three transects and three zones projected on the water's surface. A random point along the shoreline within the recognized beach area was selected to define the first transect (the leftmost transect in Figure 1). The middle transect was, then, determined as the parallel to the first transect at a distance of 20 meters, and the remaining transect, as the parallel to the middle transect at an additional distance of 20 meters, or 40 meters from the first transect. Locations at which the water attained a constant depth of 0.15, 0.5, and 1.3 meters (1.0 meters at Belle Isle, since buoys demarcating the swimming area were located at approximately this depth), corresponding to "ankle deep", "knee deep," and "chest deep" water, were selected as sampling zones. Because three of the beaches were affected by ocean tides, the actual geographic locations of these zones varied according to the tide stage. Hence, sampling locations at these beaches were fixed only in the sense that they correspond to locations at which the water depth was constant. Note the use of the term "zone" rather than "depth" in referring to areas of different water depth. This is to avoid confusion between water depth, which defines the sampling locations in Figure 1, and sampling depth, the depth below the surface of the water from which the samples were taken. In knee deep and chest water, the sampling depth was nearly always 0.3 meters from the surface, which is approximately "face deep" for a swimmer. A relatively small number of samples were taken from other sampling depths as part of a parallel study. Zones may be considered as roughly corresponding to different activities among bathers, mainly wading and infant or toddler bathing (ankle deep water), play (knee deep), and swimming or diving (chest deep).

BOOK CHAPTER The Evolution of Water Quality in the United States 1922-2003 11/01/2007
DUFOUR, A. P. AND S. SCHAUB. The Evolution of Water Quality in the United States 1922-2003. , Chapter 1, L. J. Wymer (ed.), Statistical Framework for Recreational Water Quality Criteria and Monitoring. John Wiley and Sons, LTD, , Uk, 1-12, (2007).
Abstract: The microbiological quality of recreational waters was first discussed in the United States as early as 1922 by the American Public Health Association's Committee on Bathing Beaches (APHA,1922) . The Committee surveyed 2000 physicians and state health officials inquiring about the prevalence of infections associated with bathing places. The Committee Report in 1924 (APHA,1924) reviewed the survey results and concluded that there was not enough evidence to develop bathing water standards for natural waters. In June, 1933 the Joint Committee on Bathing Places was formed and in their first report noted that because of the great lack of epidemiological information no bacterial standards were adopted (APHA, 1933). They also stated that the Committee did not want to propose arbitrary standards or measures that might promote public hysteria about the dangers of outdoor bathing places. The Committee, in 1936, was still not convinced that bathing places were a major health problem and re-stated their position on developing bacterial standards for bathing places. The reluctance to propose bacterial standards for outdoor bathing places was again evident in 1936, 1940 and 1955 (APHA, 1936, APHA, 1940, APHA, 1957). The Committee did attempt to find evidence of the risk of illness from bathing waters prior to their reports in 1940 and 1955, but they found no compelling evidence. They stated that very little reliable data were available to implicate bathing places in the spread of disease (APHA,1957).

BOOK CHAPTER The Lognormal Distribution and Use of the Geometric Mean and the Arithmetic Mean in Recreational Water Quality Measurement 11/01/2007
WYMER, L. J. AND T. J. WADE. The Lognormal Distribution and Use of the Geometric Mean and the Arithmetic Mean in Recreational Water Quality Measurement. , Chapter 6, Larry Wymer (ed.), Statistical Framework for Recreational Water Quality Criteria and Monitoring. John Wiley and Sons, LTD, , Uk, 91-112, (2007).
Abstract: Since 1968 United States recreational water quality criteria have set a limit on the geometric mean for fecal indicator bacteria from a number water samples taken over a period of time (National Technical Advisory Committee, 1968; U.S. Environmental Protection Agency, 1976 and 1986). On the other hand, for purposes of determining limits on effluents, including sewage, discharged into surface waters, the U.S. EPA specifies that calculations for all limitations which require averaging of measurements shall utilize an arithmetic mean unless otherwise specified by the Director in the permit (U.S. EPA, 1980, 2003). These limits, a geometric mean criterion for beaches and arithmetic mean for discharges, both pertain to provisions of the Clean Water Act of 1977 (CWA) as amended by the Beaches Environmental Assessment and Coastal Health (BEACH) Act of 2000. In addition to this disagreement between types of means that are used in beach monitoring and those used in limiting discharges, a trio of paper published in the late 1990's evaluated uses of the geometric mean and reached conclusions such as the use of this statistic is inappropriate for characterizing risk (Haas, 1996) and geometric means should be phased out as regulatory criteria as soon as it is practical (Parkhurst, 1998a). Statements such as these serve to further create doubt about the appropriateness of geometric means in the minds of federal and state regulators and stakeholders.
This chapter examines criticisms of the use of the geometric mean in risk assessment and environmental monitoring and evaluates its relevance to risk-based recreational water monitoring. Reasons for using the geometric mean (or rather the mean of the logarithms of the indicator densities, as we shall see) in modeling risk attributable to swimming in contaminated waters are explored and alternative models examined.

In the course of this discussion, we will refer to properties of the normal and lognormal probability distributions. For reference, a comparison of some characteristics of normal and lognormal distributions is presented in Table 1. The interested reader can find more detailed information and discussions in Crow and Shimizu (1988), Aitchison and Brown (1969) or Johnson and Kotz (1970) among other works. Estimation of lognormal parameters specifically in the context of environmental monitoring is discussed in Gilbert (1987).


BOOK CHAPTER Detection of Protozoan Parasites in Source and Finished Water 3rd Edition Asm's Methods in Environmental Microbiology 05/01/2007
SCHAEFER, F. W. Detection of Protozoan Parasites in Source and Finished Water 3rd Edition Asm's Methods in Environmental Microbiology. , 3rd., Chapter 21, Ronald L. Crawford, Jay L. Garland, David A. Lipson, Aaron L. Mills, Linda D. Stetzenbach (ed.), Methods in Environmental Microbiology. American Society for Microbiology, Washington, DC, 265-279, (2007).
Abstract: Protozoans are eukaryotic organisms which can live either a free-living or parasitic existence. Some free-living forms, under the right conditions, can become opportunistic parasites. Enteric pathogenic protozoans, like Giardia and Cryptosporidium, which are now known to be transmitted by water have been responsible for numerous waterborne outbreaks of gastroenteritis. The primary means for detection, since density levels in water are low, involves processing a large volume of water by filtration, extracting the particulates from the filter, concentrating the organisms from the particulates, and assaying for the pathogens. The most widely used method for detecting protozoans has been the indirect immunofluorescent assay. While Method 1623 has improved upon the utility of the immunofluorescent assays for Giardia and Cryptosporidium, the procedure is still labor intensive and highly dependent on the skill of the microscopist. Even with the improvements to date, this technique is known to have a number of deficiencies including false positives, inability to determine the viability and species of the detected organisms, and low average percent recovery of cysts and oocysts. Various attempts have been made to improve the immunofluorescent detection method. Rather than sampling by filtration and buoyant density centrifugation to purify the organisms, carbonate flocculation is reported to improve recoveries. PCR, cell culture, FACS, and solid phase cytometry are currently being evaluated as alternate test procedures. As each of these approaches is relatively new and much more research is needed, it remains to be seen whether they will be equal to or better than the current fluorescent assay procedure.

BOOK CHAPTER Genetic-Based Analytical Methods for Bacteria and Fungi 03/01/2007
HAUGLAND, R. A. AND S. J. VESPER. Genetic-Based Analytical Methods for Bacteria and Fungi. , Chapter 7, C.S. Yang and P. Heinsohn (ed.), Sampling and Analysis of Indoor Micoorganisms. John Wiley & Sons Incorporated, New York, NY, 133-152, (2007).
Abstract: In the past two decades, advances in high-throughput sequencing technologies have lead to a veritable explosion in the generation of nucleic acid sequence information (1). While these advances are illustrated most prominently by the successful sequencing of the human genome, they have also factored heavily in our current knowledge of nucleic acid sequences from a variety of microorganisms. Concurrent with this growth in sequencing activity has been the development of a variety of techniques for the amplification and manipulation of nucleic acids. The combination of these technologies is currently supporting a significant shift away from the use of traditional culture-based analyses for the detection and characterization of microorganisms and towards the use of new analytical methods based on genetic composition, i.e. nucleic acids, (45, 113). This shift is evident in a number of fields including the microbiological analyses of indoor environments.
Genetic analysis methods for bacterial and fungal microorganisms are presently becoming increasingly more widespread in their applications, not only in research settings, but also in clinical and environmental testing laboratories. Advantages of these methods over culture and phenotypic analyses can include higher speed, sensitivity and accuracy in the detection and identification of microorganisms as well as better resolution between similar organisms and an ability to detect non-cultivatable or fastidious organisms. Through their potential for automation, these methods also offer the possibility for less reliance on analyst training and decreased labor investments. As will be discussed in more detail below, current limitations of many of these genetic analysis methods can include on-going uncertainty of the extent to which available sequence information circumscribes the genetic variability of different target microbial groups, technical and quality control issues and higher costs related to expenditures for instruments and reagents. It is reasonable to expect, however, that each of these limitations will decrease in the future making genetic microbial testing methods an increasingly attractive option for many clinical and environmental applications.

The first section of this chapter provides an overview (with references for additional reading) of currently available genetic based analytical techniques that may be useful for investigations of bacterial and fungal microorganisms in the indoor environment. These techniques are grouped into four general categories: 1) in vitro nucleic acid amplification; 2) hybridization probes; 3) nucleic acid sequencing; and 4) microbial strain typing. The second section provides example applications of techniques within each of these categories for the study of indoor microbiology. The third section provides a synopsis of quality assurance issues and presently accepted quality control measures for laboratories performing genetic analysis methods, focusing primarily on methods involving nucleic acid amplification techniques. Other current technical limitations of these methods and their outlook for the future are also discussed.


JOURNAL Obtaining Highly Purified Toxoplasma Gondii Oocysts By a Discontinuous Cesium Chloride Gradient 11/03/2009
Staggs, S. E., M. J. See, J. Dubey, AND E. VILLEGAS. Obtaining Highly Purified Toxoplasma Gondii Oocysts By a Discontinuous Cesium Chloride Gradient. Journal of Visualized Experiments . JoVE, Somerville, MA, (33):1-2, (2009).
Abstract: Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

JOURNAL Correlation Between Ermi Values and Other Moisture and Mold Assessments of Homes in the American Healthy Home Survey 11/01/2009
VESPER, S. J., C. McKinstry, P. Ashley, D. Cox, AND G. Dewalt. Correlation Between Ermi Values and Other Moisture and Mold Assessments of Homes in the American Healthy Home Survey. JOURNAL OF URBAN HEALTH: BULLETIN OF THE NEW YORK ACADEMY OF MEDICINE. Oxford University Press, Cary, NC, 86(6):850-860, (2009).
Abstract: Objective: The objective of this study was to determine the correlation between the Environmental Relative Moldiness Index (ERMI) values in the HUD American Healthy Home Survey (AHHS) homes and either inspector reports or occupant assessments of mold and moisture. Methods: In the AHHS, moisture and mold were assessed by a trained pair of inspectors and with an occupant questionnaire. These results were compared to the ERMIresults of the Environmental Relative Moldiness Index (ERMI) values for each home. Results: Homes in the highest ERMI quartile were most often in agreement with visual inspection and/or occupant assessment. However, in 52% of the fourth quartile ERMI homes, the inspector and occupant assessment did not indicate water or mold problems. Yet the concentrations of each ERMI panel mold species detected in all fourth quartile homes were statistically indistinguishable. Conclusions: About 50% of water-damaged, moldy homes were not detected by inspection or questioning of the occupant about water and mold.

JOURNAL Quantitative Real-Time Pcr Analysis of Total Propidium Monazide-Resistant Fecal Indicator Bacteria in Wastewater 11/01/2009
VARMA, M., R. I. FIELD, M. K. STINSON, B. Rukovets, L. J. WYMER, AND R. A. HAUGLAND. Quantitative Real-Time Pcr Analysis of Total Propidium Monazide-Resistant Fecal Indicator Bacteria in Wastewater. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 43(19):4790-4801, (2009).
Abstract: A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.

JOURNAL Cryptosporidium Propidium Monoazide-Pcr, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts 11/01/2009
Brescia, C., S. Hunt, M. W. WARE, E. VARUGHESE, A. EGOROV, AND E. VILLEGAS. Cryptosporidium Propidium Monoazide-Pcr, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 75(21):6856-6863, (2009).
Abstract: Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium propidium monoazide-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to polymerase chain reaction (PCR) analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only heat-killed oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts and not dead oocysts were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof of concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical in assessing human health risks associated with this pathogen.

JOURNAL Virulence Factor-Activity Relationships: Workshop Summary 10/01/2009
DeLeon, R. Virulence Factor-Activity Relationships: Workshop Summary. JOURNAL OF WATER AND HEALTH. IWA Publishing, London, Uk, 7(S1):S94-S100, (2009).
Abstract: The concept or notion of virulence factor–activity relationships (VFAR) is an approach for identifying an analogous process to the use of qualitative structure–activity relationships (QSAR) for identifying new microbial contaminants. In QSAR, it is hypothesized that, for new chemical contaminants, their potential acute or chronic toxicity may be reasonably estimated on the basis of structural relationships to other known toxic contaminants. Thus the parallel that is being attempted for pathogenic microorganisms is that known virulence factors may be used as predictors for identifying undiscovered pathogens and microbial causes of emerging diseases. Advances in molecular biology, genomics and proteomics have led the Committee on Drinking Water Contaminants of the National Research Council, as requested by the EPA, to recommend the VFAR approach as a potentially more systematic and scientific process for the selection of microorganisms for inclusion in the Contaminant Candidate List (CCL).

JOURNAL Rooftop Runoff as a Source of Contamination: A Review 10/01/2009
LYE, D. J. Rooftop Runoff as a Source of Contamination: A Review. SCIENCE OF THE TOTAL ENVIRONMENT. Elsevier Science Ltd, New York, NY, 407(20):5429-5434, (2009).
Abstract: Scientific reports concerning chemical and microbiological contaminant levels of rainwater runoff from rooftop collection in both urban and rural areas are reviewed. This alternative source of water has been documented to often contain substantial amounts of contaminants. Studies describing levels of heavy metal contamination specific to runoff from rooftop catchment areas containing exposed metal surfaces are discussed. Depending upon the intended use, scientific evidence is also accumulating that various treatments and disinfections will be required prior to release of roof runoff water either into surface waters or for more direct consumer usage. For microbial contamination, current proposed standards and guidelines regarding this type of water source are shown to vary widely worldwide. Scientific literature reveals a lack of clarity regarding water quality guidelines and health related standards for certain types of rooftop runoff. Studies suggests that rainwater collection systems which are properly designed, maintained, and treated may provide a valuable supplement to existing water supplies by reducing demand on community water supplies/infrastructure costs, enhancing effective management of storm water runoff, and increasing restoration of underground reservoirs through controlled infiltration

JOURNAL Concentrating Toxoplasma Gondii and Cyclospora Cayetanensis from Surface Water and Drinking Water By Continuous Separation Channel Centrifugation 10/01/2009
Borchardt, M., S. Spencer, P. D. Bertz, M. W. WARE, J. P. Dubey, AND H. LINDQUIST. Concentrating Toxoplasma Gondii and Cyclospora Cayetanensis from Surface Water and Drinking Water By Continuous Separation Channel Centrifugation. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 107(4):1089-1097, (2009).
Abstract: Aims: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. Methods and Results: Ready-to-seed vials with known quantities of Toxoplasma gondii and Cyclospora cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l-1 into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were ≥84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64-100%, with the lowest recoveries in the most turbid waters. Method precision was usually between 10% and 20% coefficient of variation. Conclusion: Toxoplasma gondii and Cyclospora cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation. Significance and Impact of the Study: Waterborne transmission of Toxoplasma gondii and Cyclospora cayetanensis has been documented, presenting another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from an ambient water sample, otherwise false negatives may result. While several concentration methods are available, validation data specific to Toxoplasma gondii and Cyclospora cayetanensis recoveries are limited. Using continuous separation channel centrifugation, oocysts are recovered with high efficiency and precision, the method attributes required for accurately assessing the risk of waterborne transmission.

JOURNAL Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of C-Jun and C-Fos in Human Tissue Culture Cells 09/01/2009
HAYES, S. L., M. Waltmann, M. J. DONOHUE, D. J. LYE, AND S. J. VESPER. Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of C-Jun and C-Fos in Human Tissue Culture Cells. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 107(3):964-969, (2009).
Abstract: Aims: To assess virulence of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. Methods and Results: Aeromonas cells were added to Caco-2 cells at approximately a one to one ratio. After 1, 2 and 3 hours incubation at 37oC, mRNA was extracted from the cells and gene expression of two host genes, c-jun and c-fos, quantified. Aeromonas isolates which were virulent in the neonatal mouse model demonstrated up-regulation of c-jun and c-fos compared to avirulent isolates. Conclusions: Human cell culture results showed that c-jun and c-fos were predictive of Aeromonas virulence. Significance and Impact of the Study: An Aeromonas relative virulence scale (ARVS) is proposed for use in the testing of Aeromonas drinking water isolates.

JOURNAL Quantitative Pcr for Genetic Markers of Human Fecal Pollution 09/01/2009
SHANKS, O. C., C. A. KELTY, M. SIVAGANESAN, M. VARMA, AND R. A. HAUGLAND. Quantitative Pcr for Genetic Markers of Human Fecal Pollution. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 75(17):5507-5513, (2009).
Abstract: Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface associated genes. Both assays exhibited a range of quantification from 10 to 1x106 copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker was measured in primary effluent wastewater samples collected from 20 geographically distinct locations and compared to quantities estimated by real-time PCR assays specific for ribosomal RNA gene sequences from total Bacteroidales and enterococci fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

JOURNAL The Role of Biofilms and Protozoa in Legionella Pathogenesis: Implications for Drinking Water 08/01/2009
LAU, H. Y. AND N. ASHBOLT. The Role of Biofilms and Protozoa in Legionella Pathogenesis: Implications for Drinking Water. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 107(2):368-378, (2009).
Abstract: Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close relationship with Legionella spp. in the aquatic environment. The putative role of biofilms and amoeba in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems in controlling exposure to Legionella spp. and similar pathogens.

JOURNAL The Determination of Pesticidal and Non-Pesticidal Organotin Compounds By in Situ Ethylation and Capillary Gas Chromatography With Pulsed Flame Photometric Detection 07/01/2009
EVANS, O. M., P. KAUFFMAN, A. M. PAWLECKI-VONDERHEIDE, AND L. J. WYMER. The Determination of Pesticidal and Non-Pesticidal Organotin Compounds By in Situ Ethylation and Capillary Gas Chromatography With Pulsed Flame Photometric Detection. Microchemical Journal. Elsevier BV, AMSTERDAM, Netherlands, 92(2):155-164, (2009).
Abstract: The concurrent determination of pesticidal and non-pesticidal organotin compounds in several water matrices, using a simultaneous in situ ethylation and liquid-liquid extraction followed by splitless injection mode capillary gas chromatography with pulsed flame photometric detection, is described. The speciation analysis of nine organotin compounds includes low molecular weight - low boiling (non-pesticidal) and high molecular weight - high boiling analytes (pesticidal) of significant environmental interest. The minimum time for sodium tetraethylborate alkylation, using mechanical agitation, is determined to be fifteen minutes in order to ensure the complete derivatization of the complete list of analytes. The utilization of a “hot needle” and a rapid injection rate is shown to be an efficacious means to eliminate “mass” or “needle” discrimination when determining the mixture of organotin compounds. Method detection limits are calculated to be in the low ng L-1 range. The final method is applied to various water samples; storm water from the Cincinnati area demonstrated low native levels of three of the organotin compounds.

JOURNAL The Determination of Pesticidal and Non-Pesticidal Organotin Compounds in Water Matrices By in Situ Ethylation and Gas Chromatography With Pulsed Flame Photometric Detection 07/01/2009
EVANS, O. M., P. KAUFFMAN, A. M. PAWLECKI-VONDERHEIDE, L. J. WYMER, AND J. N. MORGAN. The Determination of Pesticidal and Non-Pesticidal Organotin Compounds in Water Matrices By in Situ Ethylation and Gas Chromatography With Pulsed Flame Photometric Detection. Microchemical Journal. Elsevier BV, AMSTERDAM, Netherlands, 92(2):155-164, (2009).
Abstract: The concurrent determination of pesticidal and non-pesticidal organotin compounds in several water matrices, using a simultaneous in situ ethylation and liquid-liquid extraction followed by splitless injection mode capillary gas chromatography with pulsed flame photometric detection, is described. The speciation analysis of nine organotin compounds includes low molecular weight - low boiling (non-pesticidal) and high molecular weight - high boiling analytes (pesticidal) of significant environmental interest. The minimum time for sodium tetraethylborate alkylation, using mechanical agitation, is determined to be fifteen minutes in order to ensure the complete derivatization of the complete list of analytes. The utilization of a “hot needle” and a rapid injection rate is shown to be an efficacious means to eliminate “mass” or “needle” discrimination when determining the mixture of organotin compounds. Method detection limits are calculated to be in the low ng L-1 range. The final method is applied to various water samples; storm water from the Cincinnati area demonstrated low native levels of three of the organotin compounds.

JOURNAL A Mouse Model for Characterization of Gastrointestinal Colonization Rates Among Environmental Aeromonas Isolates 05/01/2009
LYE, D. J. A Mouse Model for Characterization of Gastrointestinal Colonization Rates Among Environmental Aeromonas Isolates. CURRENT MICROBIOLOGY. Springer, New York, NY, 58(5):454-458, (2009).
Abstract: The colonization rates of ten different environmental isolates of Aeromonas were determined using a novel mouse-streptomycin pre-treatment method. A novel streptomycin pre-treatment prepared animals with a transient alteration in colon flora that allowed colonization by Aeromonas which would not occur in the presence of normal competitive host bacteria. Colonization rates of Aeromonas salmonicida, Aeromonas enchelia, and Aeromonas allosaccharophila were low and occurred randomly with respect to concentrations of the dosage consumed by the animals. In contrast, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae exhibited relatively high rates of colonization of mouse colon tissues.

JOURNAL Solar Radiation Disinfection of Drinking Water at Temperate Latitudes: Inactivation Rates for An Optimized Reactor Configuration 02/01/2009
Davies, C. M., D. J. Roser, A. J. Feitz, AND N. ASHBOLT. Solar Radiation Disinfection of Drinking Water at Temperate Latitudes: Inactivation Rates for An Optimized Reactor Configuration. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 43(3):643-652, (2009).
Abstract: Solar radiation-driven inactivation of bacteria, virus and protozoan pathogen models was quantified in simulated drinking water at a temperate latitude (34°S). The water was seeded with Enterococcus faecalis, Clostridium sporogenes spores, and P22 bacteriophage, each at ca 1 x 10,sup>5 mL-1, and exposed to natural sunlight in 30-L reaction vessels. Water temperature ranged from 17 to 39 °C during the experiments lasting up to 6 h. Dark controls showed little inactivation and so it was concluded that the inactivation observed was primarily driven by non-thermal processes. The optimised reactor design achieved S90 values (cumulative exposure required for 90% reduction) for the test microorganisms in the range 0.63–1.82 MJm-2of Global Solar Exposure (GSX) without the need for TiO2 as a catalyst. High turbidity (840–920 NTU) only reduced the S90 value by <40%. Further, when all S90 means were compared this decrease was not statistically significant (prob. > 0.05). However, inactivation was significantly reduced for E. faecalis and P22 when the transmittance of UV wavelengths was attenuated by water with high colour (140 PtCo units) or a suboptimally transparent reactor lid (prob. < 0.05). S90 values were consistent with those measured by other researchers (ca 1–10 MJm-2) for a range of waters and microorganisms . Although temperatures required for SODIS type pasteurization were not produced, nonthermal inactivation alone appeared to offer a viable means for reliably disinfecting low colour source waters by greater than 4 orders of magnitude on sunny days at 34°S latitude.

JOURNAL Screening Tools to Estimate Mold Burdens in Homes 01/01/2009
VESPER, S. J., C. McKinstry, K. D. BRADHAM, P. Ashley, D. Cox, G. Dewalt, AND K. Lin. Screening Tools to Estimate Mold Burdens in Homes. JOURNAL OF OCCUPATIONAL AND ENVIRONMENTAL MEDICINE. Lippincott Williams & Wilkins, Philadelphia, PA, 51(1):80-86, (2009).
Abstract: Objective: The objective of this study was to develop screening tools that could be used to estimate the mold burden in a home which would indicate whether more detailed testing might be useful. Methods: Previously, in the American Healthy Home Survey, a DNA-based method of analysis called mold specific quantitative PCR was used to measure 36 molds in standard protocol dust samples. This resulted in a national index called the Environmental Relative Moldiness Index (ERMI). In this current study, two possible screening methods were considered: use of the vacuum cleaner bag dust rather than the standard protocol dust samples and reducing the number of molds needed to be quantified resulting in the creation of a simpler mold burden scale. Results: Comparison of vacuum bag and standard dust samples from 157 of the same homes demonstrated that most molds had higher detection rates in vacuum bag dust compared to athe standard dust samples but the ERMI values were still related to each other. The second approach to simplifying the screening for mold burdens produced a correlated (ρ=0.80) index to the ERMI called the American Relative Moldiness Index (ARMI) which requires the analysis of only 12 species. Conclusions: Vacuum bag dust sample ERMI values were predictive in placing a home into the lower or upper 50% of homes on the ERMI scale. If it is not possible to obtain athe standard dust sample, the vacuum cleaner bag dust may be a useful screening tool for estimating mold burdens in homes. If the standard sample is available and a simpler screening test is sought to estimate the mold burden in homes, the ARMI scale might be useful.

JOURNAL Method Development for the Analysis of 1,4-Dioxane in Drinking Water Using Solid Phase Extraction and Gas Chromatography/Mass Spectrometry 01/01/2009
GRIMMETT, P. AND J. W. MUNCH. Method Development for the Analysis of 1,4-Dioxane in Drinking Water Using Solid Phase Extraction and Gas Chromatography/Mass Spectrometry. Journal of Chromatographic Science. Preston Publications Incorporated, Niles, IL, 47(1):31-39, (2009).
Abstract: 1,4-Dioxane has been identified as a probable human carcinogen and an emerging contaminant in drinking water. The National Exposure Research Laboratory (NERL) has developed a method for the analysis of 1,4-dioxane in drinking water at ng/L concentrations. The method consists of an activated carbon solid-phase extraction of 500-mL or 100-mL water samples using dichloromethane as the elution solvent. The extracts are analyzed by gas chromatography-mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode. In the NERL laboratory, recovery of 1,4-dioxane ranged from 94-110% in fortified laboratory reagent water (LRW), and recoveries of 96-102% were demonstrated for fortified drinking water samples. The relative standard deviations for replicate analyses were less than 6% at concentrations exceeding the minimum reporting level.

JOURNAL Review of Multi-Criteria Decision Aid for Integrated Sustainability Assessment of Urban Water Systems Mceard 12/01/2008
Lai, E., S. Lundie, AND N. ASHBOLT. Review of Multi-Criteria Decision Aid for Integrated Sustainability Assessment of Urban Water Systems Mceard. URBAN WATER. Elsevier Science Ltd, New York, NY, 5(4):315-327, (2008).
Abstract: Integrated sustainability assessment is part of a new paradigm for urban water decision making. Multi-criteria decision aid (MCDA) is an integrative framework used in urban water sustainability assessment, which has a particular focus on utilising stakeholder participation. Here MCDA is reviewed in the context of urban water management used in a decision making framework. Three other commonly used integrated approaches in urban water management (cost-benefit analysis, triple bottom line and integrated assessment) are compared with MCDA. Generic types of shortcomings associated with MCDA are discussed to provide a clear understanding of MCDA’s limitation in urban water management decision making; including 1) preferential independency, 2) double counting and under-counting, and 3) transparency of MCDA methods and results.

JOURNAL Unveiling New Degradation Intermediates/Pathways from the Photocatalytic Degradation of Microcystin-Lr 12/01/2008
Antoniou, M. G., J. A. SHOEMAKER, A. A. DELACRUZ, AND D. D. Dionysiou. Unveiling New Degradation Intermediates/Pathways from the Photocatalytic Degradation of Microcystin-Lr. ENVIRONMENTAL SCIENCE AND TECHNOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, 42(23):8877-8883, (2008).
Abstract: This study focuses on the identification of reaction intermediates formed during the photocatalytic degradation of the cyanotoxin microcystin-LR with immobilized TiO2 Tphotocatalysts at neutral pH. To differentiate between impurities already existing in the MC-LR standard (assay 96.4%) and the “true” intermediates, the peak criteria as described in a previous publication were applied (peaks must have a signal to noise ratio of 3 and twice the peak area in the treated sample when compared to initial sample) (1).

JOURNAL Mutation in the S-Ribosylhomocysteinase (Luxs) Gene Involved in Quorum Sensing Affects Biofilm Formation and Virulence in a Clinical Isolate of Aeromonas Hydrophila 11/01/2008
Kozlova, E. V., V. L. Popov, J. Sha, S. M. Foltz, T. E. Erova, S. L. Agar, A. J. Horneman, AND A. K. Chopra. Mutation in the S-Ribosylhomocysteinase (Luxs) Gene Involved in Quorum Sensing Affects Biofilm Formation and Virulence in a Clinical Isolate of Aeromonas Hydrophila. Microbial Pathogenesis. Elsevier BV, AMSTERDAM, Netherlands, 45(5-6):343-354, (2008).
Abstract: A diarrheal isolate SSU of Aeromonas hydrophila produces a cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. Our laboratory has characterized from the above Aeromonas strain, in addition to Act, the type 3- and T6-secretion systems and their effectors, as well as the genes shown to modulate the production of AI-1-like autoinducers, N-acylhomoserine lactones (AHLs) involved in quorum sensing (QS). In this study, we demonstrated the presence of an S-ribosylhomocysteinase (LuxS)-based autoinducer (AI)-2 QS system in A. hydrophila SSU and its contribution to bacterial virulence. The luxS isogenic mutant of A. hydrophila, which we prepared by marker exchange mutagenesis, showed an alteration in the dynamics and architecture of the biofilm formation, a decrease in the motility of the bacterium, and an enhanced virulence in the septicemic mouse model. Moreover, these effects of the mutation could be complemented. Enhanced production of the biofilm exopolysaccharide and filaments in the mutant strain were presumably the major causes of the observed phenotype. Our earlier studies indicated that the wild-type A. hydrophila with overproduction of DNA adenine methyltransferase (Dam) had significantly reduced motility, greater hemolytic activity associated with Act, and an enhanced ability to produce AI-1 lactones. Furthermore, such a Dam-overproducing strain was not lethal to mice. On the contrary, the luxS mutant with Dam overproduction showed an increased motility and had no effect on lactone production. In addition, the Dam-overproducing luxS mutant strain was not altered in its ability to induce lethality in a mouse model of infection when compared to the parental strain which overproduced Dam. We suggested that an altered gene expression in the luxS mutant of A. hydrophila SSU, as it related to biofilm formation and virulence, might be linked with the interruption of the bacterial metabolic pathway, specifically of methionine synthesis.

JOURNAL Cryptosporidium Source Tracking in the Potomac River Watershed Mceard 11/01/2008
Yang, W., P. Chen, E. VILLEGAS, R. B. LANDY, C. KANETSKY, V. Cama, T. Dearen, C. L. Schultz, K. G. Orndorff, G. J. Prelewicz, M. H. Brown, K. YOUNG, AND L. Xiao. Cryptosporidium Source Tracking in the Potomac River Watershed Mceard. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 74(21):6495-6504, (2008).
Abstract: To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base-flow and 28 storm-flow samples from five sites within the watershed. These sites included two water treatment plant intakes as well as three upstream sites, each associated with a different type of land use. These uses, urban/wastewater, agricultural (cattle)/wastewater, and agricultural (cattle), posed different risks with regard to the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base-flow water samples and 23 storm-flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species/genotypes, almost all wildlife-associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites under agricultural influence, it was largely absent at the urban/wastewater site. There were very few positive samples by EPA Method 1623 at any site; only eight of 90 samples (9%) analyzed were positive for Cryptosporidium by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from cattle and might not pose a significant human health risk.

JOURNAL Mold Species in Dust from the International Space Station Identified and Quantified By Mold Specific Quantitative Pcr Mceard 07/01/2008
VESPER, S. J., W. Wong, C. M. Kuo, AND D. L. Pierson. Mold Species in Dust from the International Space Station Identified and Quantified By Mold Specific Quantitative Pcr Mceard. Research in Microbiology. ELSEVIER, AMSTERDAM, Holland, 159(6):432-435, (2008).
Abstract: Dust was collected over a period of several weeks in 2007 from HEPA filters in the U.S. Laboratory Module of the International Space Station (ISS). The dust was returned on the Space Shuttle Atlantis, mixed, sieved, and the DNA was extracted. Using a DNA-based method called mold specific quantitative PCR (MSQPCR), 39 molds were measured in the dust. Potential opportunistic pathogens Aspergillus flavus and A. niger and potential moderate toxin producers Penicillium chrysogenum and P. brevicompactum were noteworthy. No cells of the potential opportunistic pathogens A. fumigatus, A. terreus, Fusarium solani or Candida albicans were detected.

JOURNAL Mold Species in Dust from the International Space Station Identified and Quantified By Mold Specific Quantitative Pcr 07/01/2008
VESPER, S. J., W. Wong, M. Kuo, AND D. L. Pierson. Mold Species in Dust from the International Space Station Identified and Quantified By Mold Specific Quantitative Pcr. Research in Microbiology. ELSEVIER, AMSTERDAM, Holland, 159(6):432-435, (2008).
Abstract: Dust was collected over a period of several weeks in 2007 from HEPA filters in the U.S. Laboratory Module of the International Space Station (ISS). The dust was returned on the Space Shuttle Atlantis, mixed, sieved, and the DNA was extracted. Using a DNA-based method called mold specific quantitative PCR (MSQPCR), 39 molds were measured in the dust. Potential opportunistic pathogens Aspergillus flavus and A. niger and potential moderate toxin producers Penicillium chrysogenum and P. brevicompactum were noteworthy. No cells of the potential opportunistic pathogens A. fumigatus, A. terreus, Fusarium solani or Candida albicans were detected.

JOURNAL Cold Shock Exoribonuclease R (Vacb) Is Involved in Aeromonas Hydrophila Pathogenesis 05/01/2008
Erova, T. E., V. G. Kosykh, A. A. Fadl, J. Sha, A. J. Horneman, AND A. K. Chopra. Cold Shock Exoribonuclease R (Vacb) Is Involved in Aeromonas Hydrophila Pathogenesis. JOURNAL OF BACTERIOLOGY. American Society for Microbiology, Washington, DC, 190(10):3467-3474, (2008).
Abstract: In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966T of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4°C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37°C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD50). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD50 dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.

JOURNAL LC/MS/MS Structure Elucidation of Reaction Intermediates Formed During the Tio2 Photocatalysis of Microcystin-Lr 05/01/2008
Antoniou, M. G., J. A. SHOEMAKER, A. A. DELACRUZ, AND D. D. Dionysiou. LC/MS/MS Structure Elucidation of Reaction Intermediates Formed During the Tio2 Photocatalysis of Microcystin-Lr. TOXICON. Elsevier Science Ltd, New York, NY, 51(6):1103-1118, (2008).
Abstract: Microcystin-LR (MC-LR), a cyanotoxin and emerging drinking water contaminant, was treated with TiO(2) photocatalysts immobilized on stainless steel plates as an alternative to nanoparticles in slurry. The reaction intermediates of MC-LR were identified with mass spectrometry (MS) at pH of Milli-Q water (pH(sq)=5.7). Eleven new [M+H](+) were observed in the liquid chromatography mass spectrometry (LC/MS) chromatogram with some of them giving multiple peaks. Most of these reaction intermediates have not been reported from previous studies employing TiO(2) nanoparicles at acidic conditions. Investigating the effects of pH, toxin adsorption and initial toxin concentration on the degradation efficiency of the TiO(2) photocatalytic firms showed that acidic conditions are preferable for the degradation. Combined with the limited surface area of the films and the absence of additional oxidants the degradation was slower and more intermediate steps were identified. Possible structures of the intermediates (formed at neutral pH) after analyzing the corresponding MS/MS spectra are reported. The collision-induced dissociation of the [M+H](+) of MC-LR and the intermediates 1011.5 and 0129.5 are discussed and possible fragmentation pathways and mechanisms are also proposed. Analysis of the MS/MS spectra indicates that the fragmentation of some amino acids is less favorable because of internal interaction with free groups of adjacent amino acids. The MS/MS spectra assisted in determining hydroxylation sites, by the formation or alteration of specific productions such as m/z 599.

JOURNAL Cold Shock Exoribonuclease R(vacb) Is Involved in Aeromonas Hydrophila Virulence 05/01/2008
Erova, T. E., V. G. Kosykh, A. A. Fadl, J. Sha, A. J. Horneman, AND A. Chopra. Cold Shock Exoribonuclease R(vacb) Is Involved in Aeromonas Hydrophila Virulence. JOURNAL OF BACTERIOLOGY. American Society for Microbiology, Washington, DC, 190(10):3467-3474, (2008).
Abstract: In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966T of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4°C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37°C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD50). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD50 dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.

JOURNAL Use of (1-3)-Β-D-Glucan Concentrations in Dust as a Surrogate Method for Estimating Specific Mold Exposures 05/01/2008
VESPER, S. J., C. McKinstry, R. A. HAUGLAND, L. M. NEAS, E. E. HUDGENS, B. HEIDENFELDER, AND J. GALLAGHER. Use of (1-3)-Β-D-Glucan Concentrations in Dust as a Surrogate Method for Estimating Specific Mold Exposures. SCIENCE OF THE TOTAL ENVIRONMENT. Elsevier Science Ltd, New York, NY, 394(1):192-196, (2008).
Abstract: Indoor exposure to fungi has been associated with respiratory symptoms, often attributed to their major cell wall component, (1-3)-β-D-glucan (DG). This and the ease and low cost of performing DG analysis rather than cultivation or microscopic counting of mold spores, has prompted many to use DG as a surrogate for mold exposure. The aim of this study was to examine which indoor mold species predict DG concentration in field dust samples, and thus whether DG can be used as a surrogate for total mold or specific mold genera exposures. We used the quantitative polymerase chain reaction (QPCR) method to analyzed 36 indoor fungal species in 297 indoor dust samples, which were also simultaneously analyzed for DG concentration. Linear regression analysis, followed by factor analysis and structural equation modeling, were utilized in order to identify fungal species that mostly contribute to the DG concentration in field dust samples. The study revealed that Cladosporium and Aspergillus species were the main DG contributors followed by Epicoccum nigrum, Wallemia sebi and Penicillium brevicompacatum. Another interesting finding of the study is that the species that contribute most to the DG concentration are also the ones that are most prevalent in indoor environments. However, Alternaria alternata, the third most common fungal species in indoor dust, did not seem to be a significant source of DG. We can speculate whether this was due to low extraction efficiency, or that allergic effects of Alternaria are not determined by the (1-3)-β-D-glucan content.

JOURNAL Higher Environmental Relative Moldiness Index (Ermism) Values Measured in Detroit Homes of Severely Asthmatic Children 05/01/2008
VESPER, S. J., C. MCKINSTRY, R. A. HAUGLAND, L. M. NEAS, E. E. HUDGENS, B. HEIDENFELDER, AND J. E. GALLAGHER. Higher Environmental Relative Moldiness Index (Ermism) Values Measured in Detroit Homes of Severely Asthmatic Children. SCIENCE OF THE TOTAL ENVIRONMENT. Elsevier Science Ltd, New York, NY, 394(1):192-196, (2008).
Abstract: Sieved vacuum bag dust from the homes of 143 children in Detroit was analyzed by mold specific quantitative PCR (MSQPCR) and the Environmental Relative Moldiness Index (ERMIsm) was calculated for each home. Children living in these homes were categorized into non-asthmatic (n=83), moderately asthmatic (n=28) and severely asthmatic (n=32) based on prescription medication usage for their asthma management (none, occasional and daily, respectively). The mean ERMI for each group of homes was 6.2 for non-asthmatic, 6.3 for moderately asthmatic and 8.2 for severely asthmatic children. The ERMI values in the homes of severely asthmatic children were significantly greater compared to the non-asthmatics (p = 0.04 in Wilcoxon Rank-sum test). Aspergillus niger and Aspergillus unguis were the primary mold species that distinguished severely asthmatic children’s homes and non-asthmatic children’s homes (p < 0.05; Wilcoxon Rank-sum test). The determination of the home’s ERMI values may aid in prioritizing home remediation efforts, particularly in those children who are at increased risk for asthma exacerbation.

JOURNAL Molecular Characterization of a Functional Type Vi Secretion System from a Clinical Isolate of Aeromonas Hydrophilia 04/01/2008
Suarez, G., J. C. Sierra, J. Sha, S. Wang, T. E. Erova, A. A. Fadl, S. M. Foltz, A. J. Horneman, AND A. K. Chopra. Molecular Characterization of a Functional Type Vi Secretion System from a Clinical Isolate of Aeromonas Hydrophilia. Microbial Pathogenesis. Elsevier BV, AMSTERDAM, Netherlands, 44(4):344-361, (2008).
Abstract: Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.

JOURNAL Complementary Molecular and Elemental Detection of Speciated Thioarsenicals Using Esi-MS in Combination With a Xenon-Based Collision-Cell ICP-MS With Application to Fortified Nist Freeze-Dried Urine 04/01/2008
ELLIS, J., C. GALLAWA, J. CARUSO, J. T. CREED, S. CONKLIN, P. A. CREED, AND A. R. YOUNG. Complementary Molecular and Elemental Detection of Speciated Thioarsenicals Using Esi-MS in Combination With a Xenon-Based Collision-Cell ICP-MS With Application to Fortified Nist Freeze-Dried Urine. Analytical and Bioanalytical Chemistry. Springer, New York, NY, 390(7):1731-1737, (2008).
Abstract: The simultaneous detection of arsenic and sulfur in thio-arsenicals was achieved using xenonbased collision cell ICP-MS in combination with HPLC. In an attempt to minimize the 16O16O+ interference at m/z 32, both sample introduction and collision cell experimental parameters were optimized. Low flow rates (0.25mL/min) and a high methanol concentration (8%) in the mobile phase produced a four fold decrease in the m/z 32 background. A plasma sampling depth change from 3mm to 7mm produced a two fold decrease in background at m/z 32, with a corresponding four fold increase in the signal associated with a high ionization surrogate for sulfur. The quadrupole and octopole bias were used as a kinetic energy discriminator between background and analyte ions, but a variety of tuning conditions produced similar (

JOURNAL Molecular Characterization of a Functional Type Vi Secretion System from a Clinical Isolate of Aeromonas Hydrophila 04/01/2008
Suarez, G., J. C. Sierra, J. Sha, S. Wang, T. E. Erova, A. A. Fadl, S. M. Foltz, A. J. Horneman, AND A. K. Chopra. Molecular Characterization of a Functional Type Vi Secretion System from a Clinical Isolate of Aeromonas Hydrophila. Microbial Pathogenesis. Elsevier BV, AMSTERDAM, Netherlands, 44(4):344-361, (2008).
Abstract: Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.

JOURNAL Tissue, Dosimetry, Metabolism and Excretion of Pentavalent and Trivalent Dimethylated Arsenic in Mice After Oral Administration 02/15/2008
HUGHES, M. F., D. Vicenta, B. ADAIR, S. CONKLIN, J. T. CREED, S. Rybb, E. M. KENYON, AND D. J. THOMAS. Tissue, Dosimetry, Metabolism and Excretion of Pentavalent and Trivalent Dimethylated Arsenic in Mice After Oral Administration. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, 227(1):26-35, (2008).
Abstract: Dimethylarsinic acid (DMA(V)) is a rat bladder carcinogen and the major urinary metabolite of administered inorganic arsenic in most mammals. This study examined the disposition of pentavalent and trivalent dimethylated arsenic inmice after acute oral administration. Adult female mice were administered [14]-DMA(V)(0.6 or 60 mg As/kg) and sacrificed serially over 24 h. Tissues and excreta were collected for analysis of radioactivity. Other mice wer administered unlabeled DMA(V)(0.6 or 60mg As/kg) or dimethylarsinous acid (DMA(III))(0.6 mg As/kg) and sacrified at 2 or 24 h. Tissues (2-h) and urine (24-h) were collected and analyzed for arsenicals. Absorption, distribution and excretion of [14C]-DMA(V) was rapid, as radioactivity was detected in tissues and urine at 0.25 h. For low dose DMA(V) mice, there was a greater fractional absorption of DMA(V) and significantly greater tissue concentrations of radioactivity at several time points. Radioactivity distributed greatest to the liver (1-2% of dose) and declined to less than 0.05% in all tissues examined after 24 h. Urinary excretion of radioactivity was significantly greater in the 0.6 mg As/kg DMA(V) group. Conversely, fecal excretion of radioactivity was significantly greater in the high dose group. Urinary metabolites of DMA(V) included DMA(III), mtrimethylarsine oxide (TMAO), dimethylthioarsinic acid and trimethylarsine sulfide. Urinary metabolites of DMA(III) included TMAO, dimethylthioarsinic acid and trimethylarsine sulfide. DMA(V) was also excreted by DMA(III)-treated mice, showing it sensitivity to oxidation. TMAO was detected in tissues of the high dose DMA(V) group. The low acute toxicity of DMA(V) in the mouse appears to be due in part to its minimal retention and rapid elimination.

JOURNAL Quantifying Fungal Viability in Air and Water Samples Using Quantitative Pcr After Treatment With Propidium Monoazide (Pma) 02/01/2008
VESPER, S. J., C. McKinstrey, C. Hartmann, M. Neace, S. Yoder, AND A. Vesper. Quantifying Fungal Viability in Air and Water Samples Using Quantitative Pcr After Treatment With Propidium Monoazide (Pma). JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 72(2):180-184, (2008).
Abstract: A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, A. flavus, A. terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85oC or held at 5oC (controls) for 1 hr. Polycarbonate filters (25 mm diameter, 0.8 µ pore size) were placed on “welled” slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light emitting diodes (LED) to inactivate remaining PMA and secure intercalation of PMA with DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate100 to1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.

JOURNAL Biological Characterization of a New Type III Secretion System Effector from a Clinical Isolate of Aeromonas Hydrophila Part II 10/01/2007
Sierra, J. C., G. Suarez, J. Sha, S. M. Foltz, V. L. Popov, C. L. Galindo, H. R. Garner, AND A. K. Chopra. Biological Characterization of a New Type III Secretion System Effector from a Clinical Isolate of Aeromonas Hydrophila Part II. Microbial Pathogenesis. Elsevier BV, AMSTERDAM, Netherlands, 43(4):147-160, (2007).
Abstract: Journal article published under Cooperative Agreement with the University of Texas

JOURNAL Further Characterization of a Type III Secretion System (T3ss) and of a New Effector Protein from a Clinical Isolate of Aeromonas Hydrophila Part I 10/01/2007
Sha, J., S. F. Wang, J. C. Sierra, A. A. Fadl, T. E. Erova, S. M. Foltz, B. K. Khajanchi, A. Silver, J. Graf, C. H. Schein, AND A. K. Chopra. Further Characterization of a Type III Secretion System (T3ss) and of a New Effector Protein from a Clinical Isolate of Aeromonas Hydrophila Part I. Microbial Pathogenesis. Elsevier BV, AMSTERDAM, Netherlands, 43(4):127-146, (2007).
Abstract: A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539 bp in length and encoded a protein of 512 amino acid (aa) residues. The NH2-terminus of AexU (aa residues 1–231) exhibited a 67% homology with the NH2-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232–512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the ΔaopB mutant, of A. hydrophila, generating a single knockout (ΔaexU) and a double knockout mutant, ΔaopB/ΔaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the ΔaopB/ΔaexU mutant, as compared to macrophages when infected with the parental ΔaopB strain. Further, mice infected with the ΔaexU mutant had a 60% survival rate, compared to animals infected with the WT or the ΔaexU-complemented strain that caused 90–100% of the animals to die at a 2–3 LD50s dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.

JOURNAL Amplified Fragment Length Polymorphism Analysis of Mycobacterium Avium Complex Isolates Recovered from Southern California 09/01/2007
PFALLER, S. L., T. C. COVERT, T. A. ARONSON, AND A. H. HOLTZMAN. Amplified Fragment Length Polymorphism Analysis of Mycobacterium Avium Complex Isolates Recovered from Southern California. Journal of Medical Microbiology. Society for General Microbiology, 56(9):1152-1160, (2007).
Abstract: Fine-scale genotyping methods are necessary in order to identify possible sources of human exposure to opportunistic pathogens belonging to the Mycobacterium avium complex (MAC). In this study, amplified fragment length polymorphism (AFLP) analysis was evaluated for fingerprinting 159 patient and environmental MAC isolates from southern California. AFLP analysis accurately identified strains belonging to M. avium and Mycobacterium intracellulare and differentiated between strains within each species. The method was also able to differentiate strains that were presumed to be genetically identical in two previous studies using large RFLP analysis with PFGE, or PCR-amplification of DNA segments located between insertion sequences IS1245 and IS1311. For M. avium, drinking-water isolates clustered more closely with each other than with patient or food isolates. Patient isolates were more genetically diverse. None of the environmental isolates shared identical AFLP patterns with patient isolates for either species. There were, however, environmental isolates that shared identical patterns, and patient isolates that shared identical patterns. A subset of the isolates, which are referred to as MX isolates due to their ambiguous identification with the Gen-Probe system, produced AFLP patterns similar to those obtained from M. intracellulare isolates. Sequence analysis of 16S rDNA obtained from the MX isolates suggests that they are strains of M. intracellulare that were not correctly identified by the M. intracellulare AccuProbe from Gen-Probe.

JOURNAL Comparison of Mold Concentrations in Indoor and Outdoor Air Sampled Simultaneously and Then Quantified By Msqpcr 08/15/2007
MEKLIN, T., T. REPONEN, C. MCKINSTRY, S. CHO, S. A. GRINSHPUN, A. NEVALAINEN, A. VEPSALAINEN, R. A. HAUGLAND, G. LEMASTERS, AND S. J. VESPER. Comparison of Mold Concentrations in Indoor and Outdoor Air Sampled Simultaneously and Then Quantified By Msqpcr. SCIENCE OF THE TOTAL ENVIRONMENT. Elsevier Science Ltd, New York, NY, 382(1):130-134, (2007).
Abstract: Mold specific quantitative PCR (MSQPCR) was used to measure the concentrations of the 36 mold species in indoor and outdoor air samples that were taken simultaneously for 48 hours in and around 17 homes in Cincinnati, Ohio. The total spore concentrations of 353 per m3 of indoor air and 827 per m3 of outdoor air samples were significantly different (p < 0.05). However, only the concentrations of Aspergillus penicillioides, Cladosporium cladosporioides types 1 and 2 and C. herbarum were correlated in indoor and outdoor air samples (p value < 0.05 and sufficient data for estimate and absolute value rho estimate > 0.5). These results suggest that interpretation of the meaning of short-term (< 48 h) mold measurements in indoor and outdoor air samples must be made with caution.

JOURNAL Opportunistic Aspergillus Pathogens Measured in Home and Hospital Tap Water By Mold Specific Quantitative Pcr (Msqpcr) 08/07/2007
VESPER, S. J., M. E. ROGERS, A. N. NEELY, AND R. A. HAUGLAND. Opportunistic Aspergillus Pathogens Measured in Home and Hospital Tap Water By Mold Specific Quantitative Pcr (Msqpcr). Charles P. Gerber (ed.), JOURNAL OF WATER AND HEALTH. IWA Publishing, London, Uk, 5(3):427-431, (2007).
Abstract: Opportunistic fungal pathogens are a concern because of the increasing number of immunocompromised patients. The goal of this research was to test a simple extraction method and rapid quantitative PCR (QPCR) measurement of the occurrence of potential pathogens, Aspergillus fumigatus, A. flavus, A. terreus and A. niger, in home tap water and a hospital water supply.
Water samples were taken from the kitchen tap in homes of 60 patients who were diagnosed with legionellosis. Water samples were also taken from three locations in a hospital that generated all of its hot water by flash heating. Opportunistic infectious agents Aspergillus fumigatus, A. flavus, A. terreus and A. niger were measured using QPCR. Aspergillus terreus DNA was found in 16.7% and A. fumigatus DNA in 1.7% of the samples taken from the kitchen tap. None of the Aspergillus species were found in any of the hospital water samples.

The development of a simple DNA extraction method along with QPCR analysis is suitable for rapid screening of tap water for opportunistic fungal pathogens. This simple method can be used to obtain pathogen occurrence results in about 3 hours; instead of waiting days to weeks for culture data. Obtaining pathogen occurrence data in a timely manner, could promote the elimination of the pathogens from the water supply of immunocompromised patients.


JOURNAL Quantitative Pcr Analysis of Molds in the Dust from Homes of Asthmatic Children in North Carolina 08/03/2007
VESPER, S. J., C. MCKINSTRY, P. ASHLEY, R. A. HAUGLAND, K. YEATTS, K. D. BRADHAM, AND E. R. SVENDSEN. Quantitative Pcr Analysis of Molds in the Dust from Homes of Asthmatic Children in North Carolina. JOURNAL OF ENVIRONMENTAL MONITORING. Royal Society of Chemistry, Cambridge, Uk, 9(8):826-830, (2007).
Abstract: The vacuum bag (VB) dust was analyzed by mold specific quantitative PCR. These results were compared to the analysis survey calculated for each of the homes. The mean and standard deviation (SD) of the ERMI values in the homes of the NC asthmatic children was 16.4 (6.77), compared to the HUD survey p = 0.003 in the NC asthmatic children's homes. The molds Chaetomium globosum, Aspergillus fumigatus, and Eurotium in the NC homes of asthmatics making the ERMI values significantly higher. Dust analysis may be useful method for estimating the mold burden in a home.

JOURNAL Development of An Environmental Relative Moldiness Index for US Homes 08/01/2007
VESPER, S. J., C. MCKINSTRY, R. A. HAUGLAND, L. J. WYMER, K. D. BRADHAM, P. ASHLEY, D. COX, G. DEWALT, AND W. FRIEDMAN. Development of An Environmental Relative Moldiness Index for US Homes. JOURNAL OF OCCUPATIONAL AND ENVIRONMENTAL MEDICINE. Lippincott Williams & Wilkins, Philadelphia, PA, 49(8):829-833, (2007).
Abstract: As part of the HUD American Healthy Homes Survey, dust samples were collected by vacuuming 2 m2 in the bedroom plus 2 m2 in the living room of a nationally representative 1096 homes in the USA using the Mitest sampler. Five mg of sieved (300 µ pore, nylon mesh) dust was analyzed by mold specific quantitative PCR for the 36 EPA Mold Panel Species. On this basis, an environmental relative moldiness index (ERMI) was created with values ranging from about -10 to 20 (lowest to highest). In order to try to reduce the cost of this analysis, the number of test species was reduced by selecting only those species with a national average concentration of 30 cell equivalents (CE) per mg dust or greater. Only 19 of 36 species met this criterion. (In 40% of the homes, an additional 46 species were quantified from the same dust sample. All of these species had average concentrations less than 30 CE per mg dust.) These 19 species were then categorized into two groups based on their coefficient of variation (CV). If the CV was > 9, the mold was placed in Category 1 (10/19) and the other molds were placed in Category 2 (9/19). Using these Categories, the sum of the log-transformed concentrations of three Category 2 molds (C. herbarum, A. alternata and C. cladosporioides Type 1) was subtracted from the sum of the log-transformed concentrations of the ten Category 1 molds (Aspergillus niger, A. ochraceus, A. penicillioides, A. restrictus, A. sydowii, Chaetomium globosum, Eurotium amsteldoami, Paecilomyces variotii, Penicillium chrysogenum and Wallemia sebi). Assembling these values for the 1096 AHHS homes from lowest to highest produced the American relative moldiness index (ARMI). The correlation between the ERMI and ARMI values was 0.88. The ERMI or ARMI scales may be useful as a standard for mold exposure estimates in epidemiological studies.

JOURNAL Tissue Distribution and Urinary Excretion of Dimethylated Arsenic and Its Metabolites in Dimethylarsinic Acid-or Arsenate-Treated Rats Mceard 07/15/2007
Adair, B. M., T. MOORE, S. CONKLIN, J. T. CREED, D. C. WOLF, AND D. J. THOMAS. Tissue Distribution and Urinary Excretion of Dimethylated Arsenic and Its Metabolites in Dimethylarsinic Acid-or Arsenate-Treated Rats Mceard. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, 222(2):235-242, (2007).
Abstract: Adult female Fisher 344 rats received drinking water containing 0, 4, 40, 100, or 200 parts per million of dimethylarsinic acid or 100 parts per million of arsenate for 14 days. Urine was collected during the last 24 h of exposure. Tissues were then taken for analysis of dimethylated and trimethylated arsenicals; urines were analyzed for these arsenicals and their thiolated derivatives. In dimethylarsinic acid-treated rats, highest concentrations of dimethylated arsenic were found in blood. In lung, liver, and kidney, concentrations of dimethylated arsenic exceeded those of trimethylated species; in urinary bladder and urine, trimethylated arsenic predominated. Dimethylthioarsinic acid and trimethylarsine sulfide were present in urine of dimethylarsinic acid-treated rats. Concentrations of dimethylated arsenicals were similar in most tissues of dimethylarsinic acidand arsenate-treated rats, including urinary bladder which is the target for dimethylarsinic acid-induced carcinogenesis in the rat. Mean concentration of dimethylated arsenic was significantly higher (Pb0.05) in urine of dimethylarsinic acid-treated rats than in arsenate-treated rats, suggesting a difference between treatment groups in the flux of dimethylated arsenic through urinary bladder. Concentrations of trimethylated arsenic concentrations were consistently higher in dimethylarsinic acid-treated rats than in arsenate-treated rats; these differences were significant (Pb0.05) in liver, urinary bladder, and urine. Concentrations of dimethylthioarsinic acid and trimethylarsine sulfide were higher in urine from dimethylarsinic acid-treated rats than from arsenate-treated rats. Dimethylarsinic acid is extensively metabolized in the rat, yielding significant concentrations of trimethylated species and of thiolated derivatives. One or more of these metabolites could be the species causing alterations of cellular function that lead to tumors in the urinary bladder.

JOURNAL Blind Trials Evaluating in Vitro Infectivity of Cryptosporidium Parvum Oocysts Using Cell Culture Immunofluorescence 05/01/2007
BUKHARI, Z., D. M. HOLT, M. W. WARE, AND F. W. SCHAEFER. Blind Trials Evaluating in Vitro Infectivity of Cryptosporidium Parvum Oocysts Using Cell Culture Immunofluorescence. CANADIAN JOURNAL OF MICROBIOLOGY. NRC Research Press, Ottawa, Canada, 53(5):656-663, (2007).
Abstract: An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspensions consisting of between 0-100% viable oocysts were prepared at the U.S. EPA, shipped to the American Water (AW) laboratory and analyzed 'blindly' by cell culture-IFA. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to a cell culture dose response curve data developed previously at AW. For test samples containing oocyst suspensions of unknown infectivity, cell culture-IFA analyses indicated a high degree of correlation (r2= 0.89; n=26) with the values expected by U.S. EPA. Cell culture infectivity correlates well with neonatal mouse infectivity assays and these 'blind' validation trials provide credibility for the cell culture-IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.

JOURNAL Characterization of a Cryptosporidium Muris Infection and Reinfection in Cf-1 Mice 03/31/2007
MILLER, T. A. Characterization of a Cryptosporidium Muris Infection and Reinfection in Cf-1 Mice. Veterinary Parasitology. Elsevier, Shannon, Ireland, 144(3-4):208-221, (2007).
Abstract: To establish control values for circulating cells and immune associated organs over the course of a self-limiting Cryptosporidium muris infection and rechallenge infection, mice were evaluated at intervals starting before oral inoculation and ending after oocyst shedding had ceased. These values were used in other experiments to evaluate changes in these parameters induced by a single dose glucocorticoid immunosuppression model and in other immunosuppression studies.

JOURNAL A Critical Evaluation of a Flow Cytometer Used for Detecting Enterococci in Recreational Waters 03/31/2007
STANG, D., K. P. BRENNER, AND M. R. RODGERS. A Critical Evaluation of a Flow Cytometer Used for Detecting Enterococci in Recreational Waters. JOURNAL OF WATER AND HEALTH. IWA Publishing, London, Uk, 5(2):295-305, (2007).
Abstract: The current U. S. Environmental Protection Agency-approved method for enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens may occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect enterococci in bathing beach waters. The flow cytometer chosen for this study was a laser microbial detection system designed to detect labeled antibodies. A comparison of MF counts with flow cytometry counts of enterococci in phosphate buffer and sterile-filtered recreational water showed good agreement between the two methods. However, when flow cytometry was used, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations. The unspiked sample controls frequently had higher counts than the samples spiked with enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody and carryover to subsequent samples. For these reasons, this technology may not be suitable for enterococci detection in recreational waters. Improvements in research and instrument design that will eliminate high background and carryover may make this a viable technology in the future.

JOURNAL Characterization of Aeromonas Virulence Using An Immunocompromised Mouse Model 03/01/2007
LYE, D. J., M. R. RODGERS, G. N. STELMA, S. J. VESPER, AND S. L. HAYES. Characterization of Aeromonas Virulence Using An Immunocompromised Mouse Model. CURRENT MICROBIOLOGY. Springer, New York, NY, 54(3):195-198, (2007).
Abstract: An immunocompromised mouse model was used to characterize Aeromonas strains for their ability to cause opportunistic, extraintestinal infections. A total of 34 isolates of Aeromonas (A. hydrophila [n = 12]), A. veronii biotype sobria [n = 7], A. caviae [n = 4], A. enchelia [n = 4], A. allosaccharophila [n = 2], A. salmonicida (n = 4), and A. bestiarum [n = 1]) were introduced by intraperitoneal injection into immunocompetent or chemically compromised (using cyclophosphamide) mice. The ability of each isolate to persist in the liver and spleen tissue was monitored at 24 hours after exposure.Amajority of A. hydrophila and A veronii v. sobria strains, but none of the isolates of other Aeromonas species, were capable of persistent colonization (<300 cells/mg spleen and liver tissue at 24 hours). The presence or absence of several putative virulence factors (cytotoxicity to HEp-2, lipase activity, elastase activity, and hemolysis) were determined for each isolate using in vitro tests. There were no correlations between the presence or absence of biochemical test results for putative virulence factors and persistence of the isolate in spleen and liver tissue at 24 hours.

JOURNAL Differentiation of Aeromonas Isolates Obtained from Drinking Water Distribution System Using Matrix-Assisted Laser Description/Ionization-Mass Spectrometry (Maldi-MS) 02/02/2007
DONOHUE, M. J., J. BEST, W. SMALLWOOD, M. KOSTICH, M. R. RODGERS, AND J. A. SHOEMAKER. Differentiation of Aeromonas Isolates Obtained from Drinking Water Distribution System Using Matrix-Assisted Laser Description/Ionization-Mass Spectrometry (Maldi-MS). Analytical Chemistry. American Chemical Society, Washington, DC, 79(5):1939-1946, (2007).
Abstract: The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to analyze the whole cells of both reference strains and unknown Aeromonas isolates obtained from water distribution systems. A library of over 45 unizue m/z signatures was created from 40 strains that are representative of the seventeen recognized species of Aeromonas, as well as three reference strains from genus Vibrio and two reference strains from Plesiomonas shigelloides. The library was used to help speciate 52 isolates of Aeromonas. The environmental isolates were broken up into two blind studies. Group 1 contained isolates that had a recognizable phenotypic profile and Group 2 contained isolates that had an atypical phenotypic profile. MALDI-MS analysis of the water isolates in Group 1 matched the phenotypic identification in all cases. In Group 2, the MALDI-MS based determination confirmed the identity of 18 of the 27 isolates. These results demonstrate the MALDI-MS analysis can rapidly and accurately classify species of the genus Aeromonas, making it a powerful tool especially suited for environmental monitoring and detection of microbial hazards in drinking water.

JOURNAL Importance of Flagella and Enterotoxins for Aeromonas Virulence in a Mouse Model 02/01/2007
SEN, K. AND D. J. LYE. Importance of Flagella and Enterotoxins for Aeromonas Virulence in a Mouse Model. CANADIAN JOURNAL OF MICROBIOLOGY. NRC Research Press, Ottawa, Canada, 53(2):261-269, (2007).
Abstract: A genetic characterization of eight virulence factor genes, elastase, lipase, polar flagella (flaA/flaB, flaG), lateral flagella (lafA), and the enterotoxins alt, act, and ast, was performed using polymerase chain reaction with 55 drinking water and nine clinical isolates. When 16 Aeromonas hydrophila strains, seven Aeromonas veronii strains, and seven Aeromonas caviae strains exhibiting different combinations of virulence factor genes were tested in immunocompromised mice by intraperitoneal injection, only those strains that had one or more of the enterotoxins flaA, flaB, and either flaG or lafA showed signs of being virulent. The correlation was seen in 97% (29/30) of the strains, which included strains from drinking water. Thus, Aeromonas water isolates have the potential to be pathogenic in immunocompromised hosts.

JOURNAL Speciation of Organotins in Poly Vinyl Pipe Via X-Ray Absorption Spectroscopy and in Leachates By Elthylation/Derivitization 02/01/2007
IMPELLITTERI, CHRISTOPHER, O. M. EVANS, AND B. RAVEL. Speciation of Organotins in Poly Vinyl Pipe Via X-Ray Absorption Spectroscopy and in Leachates By Elthylation/Derivitization. JOURNAL OF ENVIRONMENTAL MONITORING. Royal Society of Chemistry, Cambridge, Uk, 9(4):358-365, (2007).
Abstract: Three different polyvinyl chloride (PVC) pipe types were subjected to de-ionized water exposures over the course of at least 180 days. Water exposed to the pipe was analyzed for organotin speciation and concentration. Organotin concentrations were the highest during the first 1-5 days. The species and concentrations of organotins leached varied by pipe type. Data were normalized by surface area in order to compare laboratory results with results from a residential pipe system. For one pipe type, the lowest non-zero concentrations from the laboratory tests overestimated organotin concentrations in solution when compared with water samples from the same pipe type in a residence. For organotin exposure estimates, a range of 0.1 ng mµ 2 to 10 ng mµ 2 could be used for mature pipes (e.g. in use for 1 year). These estimates should be refined with more field study, however, due to the high variation in organotin species and concentrations leached as a function of pipe type, accuracy within an order of magnitude may be optimal as, in many instances, the type of pipe installed or buried may be unknown. X-ray absorption spectroscopy (XAS) was used to identify organic and inorganic tin species in reference materials and the PVC samples. Monobutyl tin was identified as the primary organotin species in the pipes. Results from the XAS analyses also indicate that the technique shows promise for distinguishing between inorganic tin and organotins. Furthermore, organotins may be distinguished between mono-, di-, and tri-ligand species using XAS.

JOURNAL Dimethylthioarsinic Anhydride: A Standard for Arsenic Speciation 01/30/2007
FRICKE, M., M. ZELLER, W. R. CULLEN, M. R. WITKOWSKI, AND JOHN T. CREED. Dimethylthioarsinic Anhydride: A Standard for Arsenic Speciation. ANALYTICA CHIMICA ACTA. Elsevier Science Ltd, New York, NY, 583(1):78-83, (2007).
Abstract: Dimethylthioarsinic acid (DMTAV) has recently been identified in biological, dietary and environmental matrices. The relevance of this compound to the toxicity of arsenic in humans is unknown and further exposure assessment and metabolic studies are difficult to conduct because of the unavailability of a well characterized standard. The synthesis of DMTAV was accomplished by the reaction of dimethylarsinic acid (DMAV) with hydrogen sulfide. The initial reaction product produced is DMTAV but multiple products over the course of the reaction are also observed. Therefore, a chromatographic separation was developed to monitor the reaction progress via LC ICP MS. In this synthesis, conversion of DMAV to DMTAV was not taken to completion to avoid the production of side products. The product was isolated from the starting material by standard organic techniques. Single crystal diffraction demonstrated that solid DMTAV is present in the form of the oxygen-bridged dimethylthioarsinic anhydride. Dissolution of the anhydride in water produces the acid form of DMTAV and the aqueous phase DMTAV provided a characteristic molecular ion of m/z 155 by LC ESI-MS. The synthesis and isolation of dimethylthioarsinic anhydride provides a stable crystalline standard suitable for identification, toxicological study and exposure assessment of dimethylthioarsinic acid.

JOURNAL Photocatalytic Tio2 Films and Membranes for the Development of Efficient Wastewater Treatment and Reuse Systems 01/05/2007
CHOI, H., M. G. ANTONIOU, A. A. DELACRUZ, E. STATHATOS, AND D. D. DIONYSIOU. Photocatalytic Tio2 Films and Membranes for the Development of Efficient Wastewater Treatment and Reuse Systems. DESALINATION. Elsevier Science Ltd, New York, NY, 202(1-3):199-206, (2006).
Abstract: In order to develop efficient photocatalytic TiO2 films and membranes for application in water and wastewater treatment and reuse systems, there is a great need to tailor-design the structural properties of TiO2 material and enhance its photocatalytic activity. Through a simple sol-gel route, employing self-assembled surfactant molecules as pore directing agents along with acetic acid-based sol-gel route, we have fabricated nanostructured crystalline TiO2 thin films and TiO2/Al2O3 composite membranes with simultaneous photocatalytic, disinfection, separation, and anti-biofouling properties. The highly porous TiO2 material exhibited high specific surface area and porosity,narrow pore size distribution, homogeneity without cracks and pinholes, active anatase crystal phase, and small crystallite size. These TiO2 materials were highly efficient in the decomposition of methylene blue dye and creatinine, destruction of biological toxins (microcystin-LR), and inactivation of pathogenic microorganisms (Escherichia coli). Moreover, the photocatalytic TiO2 membranes exhibited not only high water permeability and sharp polyethylene glycol retention but also less adsorption fouling tendency. Here, we report results on the synthesis, characterization, and environmental application and implication of photocatalytic TiO2 films and membranes.

JOURNAL Changes in Mouse Cirulating Leukocyte Numbers in C57bl/6 Mice Immunosuppressed for Cryptosporidium Parvum Oocyst Production 01/01/2007
Miller, T A. AND F W. Schaefer III. Changes in Mouse Cirulating Leukocyte Numbers in C57bl/6 Mice Immunosuppressed for Cryptosporidium Parvum Oocyst Production. Veterinary Parasitology. Elsevier, Shannon, Ireland, 143:99-105, (2007).
Abstract: The Iowa strain of Cryptosporidium parvum will not propagate in immunocompetent mice, but will successfully infect genetically immunocompromised Nude or SCID mice as well as immunocompetent mice which have been immunosuppressed with glucocorticoids. Using dexamethasone - tetracycline is one method for immunosuppressing mice for the production of C. parvum oocysts. However, dexamethasone induced immunosuppression is variable, because it is dependent on the total daily water consumption of each individual mouse. In an attempt to more accurately characterize the immunocompromised state for future studies on the infectivity of Cryptosporidium in immunocompromised mice, the changes in circulating leukocytes and other immune system associated organs before, during and after dexamethasone suppression were analyzed. The dexamethasone induced immunocompromised state was associated with greater than 90 percent sustained drop in the circulating T-lymphocyte count, a greater than 700 percent increase in circulating mature segmented neutrophils and a severe depletion of circulating monocytes. The thymus and spleen decreased in size by over 80 percent. Oocyst shedding in suppressed mice started within four days of oocyst inoculation and persisted for six days after dexamethasone withdrawal. Circulating neutrophils rose dramatically by 714 percent while on dexamethasone; seven days after dexamethasone withdrawal circulating neutrophils still were 549 percent higher than normal. Circulating CD3 and CD4 lymphocytes remained 85 to 90 percent below normal while on dexamethasone and for seven days after discontinuing dexamethasone. CD8 lymphocyte numbers initially decreased by 90 percent, but rose even while on dexamethasone and even with severe thymic involution. At day seven post dexamethasone treatment, the spleen was 119 mm3, approximating normal. After fourteen days of dexamethasone withdrawal, the CD8 counts were only 1.6 percent below normal while the CD3 and CD4 counts were still 66 percent below normal. The thymus now was about three quarters of its normal size. The rise in circulating CD8 lymphocytes when oocyst production stopped suggests that CD8 positive lymphocytes may play a significant role in vivo in clearing the parasite.

JOURNAL Relative Moldiness Index© as Predictor of Childhood Respiratory Illness 01/01/2007
VESPER, S. J., C. MCKINSTRY, R. A. HAUGLAND, Y. IOSSIFOVA, G. LEMASTERS, L. LEVIN, G. K. HERSHEY, M. VILLAREAL, D. I. BERNSTEIN, J. LOCKEY, AND T. REPONEN. Relative Moldiness Index© as Predictor of Childhood Respiratory Illness. Journal of Exposure Science and Environmental Epidemiology . Nature Publishing Group, London, Uk, 17(1):88-94, (2007).
Abstract: The results of a traditional visual mold inspection were compared to a mold evaluation based on the Relative Moldiness Index (RMI). The RMI is calculated from mold specific quantitative PCR (MSQPCR) measurements of the concentation of 36 species of molds in floor dust samples. These two prospective mold evaluations were used to classify the mold condition in 271 homes of infants. Later, the development of respiratory illness was measured in the infants living in these homes and the predictive value of each classification system evaluated. The binary classification of homes as either moldy or non-moldy by on-site vidual home inspection was not predictive of the development of respiratory illness (wheeze and/or rhinitis) (p=0.27). Conversely, a method developed and validated in this paper using the RMI index fit to a logistic function, can be used to predict the occurrence of illness in homes and allows stakeholders the choice among various levels of risk.

PRESENTATION Water Sample Filtration Methods Using Viradel Procedures 11/15/2009
CASHDOLLAR, J. Water Sample Filtration Methods Using Viradel Procedures. Presented at Water Quality Technology Conference, Seattle, WA, November 15 - 19, 2009.
Abstract: To be presented at WQTC, November 15-19, Seattle, Washington

PRESENTATION Quantitative Microbial Risk Assessment (Qmra) as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches 11/09/2009
SCHOEN, M. E. AND N. ASHBOLT. Quantitative Microbial Risk Assessment (Qmra) as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches. Presented at 137th Annual APHA Meeting , Philadelphia, PA, November 09 - 11, 2009.
Abstract: The US EPA and WHO have set recreational water quality standards based on epidemiologic studies to protect human health at beaches. These studies have largely been limited to sewage-impacted sites and resources are unlikely to be available to assess the myriad of other impacted sites. Here we describe how quantitative microbial risk assessment (QMRA) can be used to assess unstudied pathogen sources in a systematic way to describe risk uncertainty. To illustrate the proposed QMRA comparison an illustrative example is provided focusing on the non-sewage example sources of seagulls and human shedders.

PRESENTATION Qmra as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches 11/07/2009
SCHOEN, M. E. Qmra as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches. Presented at American Public Health Association Annual Meeting, Philadelphia, PA, November 07 - 11, 2009.
Abstract: The US EPA and WHO have set recreational water quality standards based on epidemiologic studies to protect human health at beaches. These studies have largely been limited to sewage-impacted sites and resources are unlikely to be available to assess the myriad of other impacted sites. Here we describe how quantitative microbial risk assessment (QMRA) can be used to assess unstudied pathogen sources in a systematic way to describe risk uncertainty. A QMRA was constructed based on a recreational beach primarily impacted by seagull feces, assumed to contribute only Campylobacter and Salmonella. The pathogen dose distribution was derived from the concentration of the fecal indicator in the water column using an uncertain ratio of indicator to pathogen concentrations. The probability of gastrointenteritis was calculated using dose-response relationships from the literature. All uncertain model parameters were represented by probability distributions and sampled in a Monte Carlo analysis, allowing subsequent parameter importance analysis. Based on the high uncertainty of human-infectious pathogens in gull feces, the predicted probability of infection from gulls is of potential concern when the water quality indicator is near the single sample water quality standard (104 enterococci / 100 mL). This pathogen uncertainty is common with many animal sources of fecal contamination; hence, it is important that future research focus on specifying pathogen densities to allow comparison of risk estimates from epidemiologic studies with QMRA, which may ultimately allow risk characterization from unstudied sources of fecal contamination at recreational beaches.

PRESENTATION Detection of Quantification of Mycobacterium Avium Complex Organisms in Drinking Water 11/03/2009
PFALLER, S. L. Detection of Quantification of Mycobacterium Avium Complex Organisms in Drinking Water. Presented at International Society for Exposure Science, Minneapolis, MN, November 01 - 05, 2009.
Abstract: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U. S. Environmental Protection Agency’s Contaminant Candidate List 2 (CCL2) due to their association with human disease and occurrence in public drinking water systems. Current methods for detecting MAC organisms in drinking water are culture-based. However evidence suggests that culture-based methods have severe limitations including long incubation periods, loss of target due to overgrowth of background organisms, up to 70% loss of target due to harsh decontamination techniques, and inability to recover MAC in a viable-but-non-culturable state. Because of these drawbacks and the need for more accurate and comprehensive occurrence data, we have developed real-time QPCR assays for detection and quantification of MA, MI, and MA subsp. paratuberculosis (MAP) in drinking water. Real-time QPCR assays can be used in combination with culture-based methods in order to confirm viability of MAC in samples, as well as provide isolates for further characterization. We are currently evaluating these methods for use on actual drinking water samples in order to generate a more complete understanding of MAC occurrence in drinking water.

PRESENTATION Surveillance Systems for Waterborne Protozoa Past, Present and Future 11/01/2009
VILLEGAS, E. Surveillance Systems for Waterborne Protozoa Past, Present and Future. Presented at International Society of Exposure Sciences, Minneapolis, MN, November 01 - 05, 2009.
Abstract: OVERVIEW I. Brief introduction to waterborne Cryptosporidium Historical perspective on detecting Cryptosporidium Current detection methodologies II. US EPA’s waterborne protozoan research program Detecting, typing, and tracking sources of Cryptosporidium contamination III. The future of the “Microbial Detection Toolbox”

PRESENTATION Using Propidium Monoazide as An Enrichment Step to Remove Extraneous Dma and Non-Viable Organisms for Cryptosporidium Genotyping 10/05/2009
VILLEGAS, E. Using Propidium Monoazide as An Enrichment Step to Remove Extraneous Dma and Non-Viable Organisms for Cryptosporidium Genotyping. Presented at Global Conference on Waterborne Microbial Contaminants, Singapore, SINGAPORE, October 05 - 08, 2009.
Abstract: Cryptosporidium is an important protozoan parasite that continues to cause waterborne disease outbreaks worldwide. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium propidium monoazide-polymerase chain reaction (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis. The purpose of the treatment was to prevent the amplification of DNA that was extraneous or in inactivated oocysts. Our results demonstrated that PMA effectively removes naked DNA present in environmental samples. In addition, this CryptoPMA-PCR assay can specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, only live oocysts were detected in raw waste or surface water samples that were spiked with either live or dead Cryptosporidium oocysts. This proof of concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical in assessing human health risks associated with this pathogen.

PRESENTATION The Birds Did It! 09/29/2009
SCHOEN, M. E. AND N. ASHBOLT. The Birds Did It! Presented at Great Lakes Beach Association Conference, Milwaukee, WI, September 28 - October 01, 2009.
Abstract: The US EPA and WHO have set recreational water quality standards based on epidemiologic studies to protect human health at beaches. These studies have largely been limited to sewage-impacted sites, and resources are unlikely to be available to assess the myriad of other impacted sites. Here we describe how quantitative microbial risk assessment (QMRA) can be used to assess unstudied pathogen sources in a systematic way to describe risk uncertainty. A QMRA was constructed based on a recreational beach primarily impacted by seagull feces, assumed to contribute only Campylobacter and Salmonella. The pathogen dose distribution was derived from the concentration of the fecal indicator in the water column using an uncertain ratio of indicator to pathogen concentrations. The probability of gastrointenteritis was calculated using dose-response relationships from the literature. All uncertain model parameters were represented by probability distributions and sampled in a Monte Carlo analysis, allowing subsequent parameter importance analysis. Based on the high uncertainty of human-infectious pathogens in gull feces, the predicted probability of infection from gulls is of potential concern when the water quality indicator is near the single sample water quality standard (104 enterococci / 100 mL). This pathogen uncertainty is common with many animal sources of fecal contamination; hence, it is important that future research focus on specifying pathogen densities to allow comparison of risk estimates from epidemiologic studies with QMRA, which may ultimately allow risk characterization from unstudied sources of fecal contamination at recreational beaches.

PRESENTATION Contaminants of Emerging Concerns 08/20/2009
GLASSMEYER, S., E. T. Furlong, D. W. Kolpin, R. J. MILTNER, AND S. L. Werner. Contaminants of Emerging Concerns. Presented at ASIWPCA Webinar, Cincinnati, OH, August 20, 2009.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-ug/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. To determine which wastewater chemicals persist through drinking water treatment, a follow-up study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 84 different emerging contaminants, including 24 pharmaceuticals. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-five chemicals were detected at least once, with 28 chemicals detected in the source waters and 23 chemicals detected in the finished waters. The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Peak Health and the Need for More Sustainable Urban Water Systems 08/18/2009
ASHBOLT, N. Peak Health and the Need for More Sustainable Urban Water Systems. Presented at World City Water Forum 2009, Incheon, SOUTH KOREA, August 18 - 21, 2009.
Abstract: Large centralized urban water services in developed countries like the USA still provide significant environmental impact via loss of ecological water services, energy use, loss of nutrients from agricultural production, and eutrophication issues. Current climate models predict that many regions will generally be increasingly more water stressed as well as prone to intense storm events, further exacerbating the negative effects of centralized water services. As a consequence of many interacting factors based around energy/water use and social inequity, Peak Health may have already been reached in the USA, with life expectancies equal to that of Cuba (75 years). From a global perspective, rapidly developing regions, most of which are in water scarce regions, make water-based sanitation unsustainable if not impractical. Hence there is a need to rethink how water services can be obtained for the health of developed and developing regions by lowering our environmental footprint as well as empowering individuals/communities to control their water/sanitation services in a health-promoting environment. Examples include net energy production from organic components along with nutrients returned to agriculture, particularly phosphorus that has known stores of available rock phosphate to only last 60-150 years. From a public health perspective, aging water mains and their vulnerability to intrusions by fecally-contaminated waters is a rising issue; all the more reason to consider an alternative approach to water distribution and handling of associated wastewater streams rather than rebuilding more of the same problem. Most interesting is that the single largest cause of waterborne illness identified in the US (legionellosis) is not of fecal origin, nor currently regulated in most parts of the world. Legionellosis is due to the growth of a pathogen (Legionella pneumophila), which may largely be an in-premise (building) issue rather than the distribution system per se. Hence Legionella and other similar indigenous pathogens that grow in pipe biofilms are not necessarily the responsibility of the distribution system provider. As we move to greater reliance on reclaimed waters, fit-for-purpose, the long-term ramifications of fecal and indigenous pathogen issues should be considered within a broader sustainability assessment if we are to further improve public health. A framework for a way forward is described.

PRESENTATION Community-Scale and Household-Scale Decentralized Reuse Experiences on Two Continents 07/26/2009
ASHBOLT, N. Community-Scale and Household-Scale Decentralized Reuse Experiences on Two Continents. Presented at Decentralized Water Systems Workshop, Nags Head, NC, July 26 - 29, 2009.
Abstract: Slide presentation

PRESENTATION Environmental Perspectives and Issues 07/26/2009
ASHBOLT, N., R. BASTIAN, AND R. GOO. Environmental Perspectives and Issues. Presented at Decentralized Water Systems Workshop, Nags Head, NC, July 26 - 29, 2009.
Abstract: Slide presentation

PRESENTATION Population Based Exposure Assessment of Bioaccessible Arsenic in Carrots 06/07/2009
Yathavakilla, S. K., A. R. Young, S. E. Lenhof, M. Mantha, C. GALLAWA, P. A. CREED, J. XUE, AND J. T. CREED. Population Based Exposure Assessment of Bioaccessible Arsenic in Carrots. Presented at International Symposium on Mettalomics, Covington, KY, June 07 - 13, 2009.
Abstract: The two predominant arsenic exposure routes are food and water. Estimating the risk from dietary exposures is complicated, owing to the chemical form dependent toxicity of arsenic and the diversity of arsenicals present in dietary matrices. Two aspects of assessing dietary exposure risk, which are often overlooked in speciation analysis, are producing a bioaccessibility estimate and the need to collect samples which support a population based exposure assessment. In an attempt to address these shortcomings, the authors have applied an enzymatic extraction to carrots which were collected from various geographical locations based on harvest demographics. The extracts were speciated by IC-ICP-MS utilizing collision cell technology to address the high chloride concentrations characteristic of in-vitro assays designed to mimic the gastrointestinal tract. The distribution of the inorganic arsenic concentration in carrots was then combined with the distribution associated with carrot consumption in the US using a probabilistic based model. The model randomly selects an arsenic concentration and consumption rate from the distributions and through an iterative process generates a population based exposure profile for arsenic in carrots. This approach to population based exposure assessment is especially applicable to arsenic because over 90% of the exposure can be attributed to 4-5 commonly consumed foods. This limited number of foods produces a relatively robust estimate without having to independently estimate the contribution of all other foods to the cumulative exposure. This presentation will discuss the limitations associated with the IC-ICP-MS approach and outline the application of the model to this type of exposure assessment.

PRESENTATION Analysis of Arsenicals and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS 06/07/2009
KUBACHKA, K., C. GALLAWA, P. A. CREED, J. T. CREED, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Analysis of Arsenicals and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS. Presented at International Symposium on Metallomics, Covington, KY, June 07 - 13, 2009.
Abstract: Recent metabolic and exposure assessment studies have found sulfur analogs (thioarsenicals) of common oxoarsenicals in environmental and biological systems. The occurrence of thioarsenicals raises questions regarding their origin and transport, and their roles in metabolism of arsenic. Definitive answers to these questions require speciation based methods that can separate and quantify oxo- and thioarsenicals in complex mixtures. Various LC methods are often necessary to separate such complex mixtures. Because thioarsenicals are often present in samples at low ng/g concentrations, elemental detection via ICP-MS is most commonly used for quantitation. Additionally, LC-ESI-MS/MS techniques have proven valuable for complementary verification of standards and confirmation of suspect peaks, because many arsenic standards and their sulfur analogs are not commercially available. An emerging area of arsenic metabolic research focuses on understanding of origin of thioarsenicals that are detected as excreted metabolites in many higher organisms, including humans. We have used an in vitro assay to examine the role of the anaerobic microbiota of mouse cecum in the metabolism of thio- and oxoarsenicals1-3. These studies have shown that there is considerable preabsorption metabolism of these arsenicals that is mediated by the anaerobic microbiota of mouse cecum. This presentation will summarize results from our studies on the conversion of dimethylarsinic acid and trimethylarsine oxide with an emphasis on utilizing ESI-MS/MS for identification. In addition, preliminary research on the role of the anaerobic microbiota in conversion of inorganic arsenic and monomethylarsonic acid to thioarsenicals will be presented.

PRESENTATION Overview of Challenges for Water Quality Protection and Conservation Across the Food Supply Chain and Tools for Assessing Sustainability 06/06/2009
ASHBOLT, N. Overview of Challenges for Water Quality Protection and Conservation Across the Food Supply Chain and Tools for Assessing Sustainability. Presented at IFT Conference, Anaheim, CA, June 06 - 09, 2009.
Abstract: To be presented at the IFT Conference in Anaheim, CA, 06/6-9/2009

PRESENTATION Role of Waterborne Pathogens in the Food Supply Chain: Implications to Risk Management With Local and Global Perspectives 06/06/2009
ASHBOLT, N. Role of Waterborne Pathogens in the Food Supply Chain: Implications to Risk Management With Local and Global Perspectives. Presented at IFT Conference, Anaheim, CA, June 06 - 09, 2009.
Abstract: Microbial risk assessment (MRA) in the food industry is used to support HACCP – which largely focuses on bacterial pathogen control in processing foodstuffs 􀂴 Potential role of microbially-contaminated water used in food production is not as well understood 􀂴 Emergence of Quantitative MRA as a tool for assessing & informing waterborne pathogen risks internationally WHO: water safety plans (2004), wastewater reuse (2006) 􀂴 Primary point of this paper is to highlight potential risks from water in food production 1

PRESENTATION Diversity of Free-Living Amoebae in a Dual Distribution (Potable and Recycled) Water System 06/01/2009
Thomas, J., M. Storey, R. Stuetz, S. Kjelleberg, AND N. ASHBOLT. Diversity of Free-Living Amoebae in a Dual Distribution (Potable and Recycled) Water System. Presented at IWA Health-Related Water Microbiology Biannual Conference, Naxos, GREECE, June 01 - 04, 2009.
Abstract: Free-living amoebae are known to facilitate the growth of water associated pathogens. This study, for the first time, explored the diversity of free-living amoebae in a dual distribution (potable and recycled) water system in Rouse Hill NSW, Australia. Water and biofilm samples were taken from the tertiary recycled water treatment plant and from within the two distribution systems (using a Modified Robbins Device). Amoebae were not detected entering the distribution systems from the tertiary recycled water treatment plant over a 6 week sampling period. However, amoebae were isolated within the recycled water distribution system (0.1 amoebae/ml). Furthermore, amoebae were isolated from within the potable water distribution system (0.2 amoebae/ml). Chlorine concentrations appear to explain the different amoebae numbers. More research is required to determine the relationship between these free-living amoebae and water associated pathogens within this water distribution system.

PRESENTATION Molecular-Based Detection Systems for Cryptosporidium Oocysts 05/20/2009
VILLEGAS, E. Molecular-Based Detection Systems for Cryptosporidium Oocysts. Presented at 2009 USEPA STAR Grants Workshop on Innovative Approaches to Detecting Microorganisms and Cyanotoxins in Water, Philadelphia, PA, May 20 - 21, 2009.
Abstract: The presentation describes on-going studies in collaboration with US EPA Region 2, 3, and the CDC on identifying sources of Cryptosporidium oocyst contamination in source waters using conventional and real-time PCR approaches.

PRESENTATION The Virtual Environmental Microbiology Center a Social Network for Enhanced Communication Between Water Researchers and Policy Makers 05/17/2009
Mistry, J. H., F. S. HAUCHMAN, M. R. RODGERS, AND N. ASHBOLT. The Virtual Environmental Microbiology Center a Social Network for Enhanced Communication Between Water Researchers and Policy Makers. Presented at American Society for Microbiology 109th General Meeting, Philadelphia, PA, May 17 - 21, 2009.
Abstract: Effective communication within and between organizations involved in research and policy making activities is essential. Sharing information across organizational and geographic boundaries can also facilitate coordination and collaboration, promote a better understanding of technical and policy issues, and avoid duplication of effort. To enhance communication within the environmental microbiology community, the U.S. Environmental Protection Agency (EPA) developed the Environmental Science Connector (ESC) (http://portal.epa.gov/ESC ), a web-based communication tool that can bring together people with diverse interests and expertise to share information on a wide range of environmental microbiology topics. It also provides a convenient means for EPA scientists to manage projects, interact with project collaborators through message boards, and participate in real-time web based seminars. The ESC is being used to bring the environmental microbiology community together to share information on a variety of topics. Using the ESC as a platform, a virtual seminar series has been launched to allow subject matter experts in the fields of environmental microbiology and microbial ecology to openly discuss state-of-the-science research topics with network members, further fostering a sense of community and exchange of ideas. In addition, the ESC is providing a focal point for intra-governmental communication and collaboration on several specific research topics, including sample preparation for waterborne pathogens, the development of graywater guidance and microbial risk assessment.

PRESENTATION Utilization of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (Maldi-MS) to Identify Environmental Strains of Mycobacterium Complex 05/17/2009
DONOHUE, M. J., J. H. Mistry, AND S. L. PFALLER. Utilization of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (Maldi-MS) to Identify Environmental Strains of Mycobacterium Complex. Presented at American Society for Microbiology Annual Meeting, Philadelphia, PA, May 17 - 21, 2009.
Abstract: Species within the Mycobacterium avium Complex (MAC) group are found to be both prevalent and persistent in drinking water distribution systems. The MAC is composed of two predominant species: M. avium and M. intracellulare. These species have the ability to survive drinking water disinfection treatments and to thrive in distribution systems via biofilms. For these reasons, the US Environmental Protection Agency (EPA) has listed MAC on the Contaminant Candidate List (CCL) as a potential contaminant that may require regulation if it is proven to have a significant health burden to the public. A major challenge associated with environmental MAC isolates is the ability to rapidly identify the isolate to the species level. The tools currently available for identification/speciation can be time-consuming, as well as give ambiguous results due to their inability to clearly differentiate species. The purpose of this study was to evaluate Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) as a means to rapidly speciate MAC environmental isolates. The research presented will demonstrate the use of MALDI-MS to speciate environmental isolates based on their m/z signature. Initially, a database of m/z signatures was constructed using MAC reference and type strains. Next, m/z signatures from environmental isolates were compared to the database of known m/z signatures. Additionally, DNA sequence analyses of genes coded for the heat shock protein 65 (hsp65), RNA polymerase subunit B (rpoB), and 16S rRNA were used to validate the MALDI-MS determination. MALDI-MS analysis is ideal for the identification/speciation of environmental isolates. This is primarily due to the minimal sample preparation involved (i.e., the ability to go from culture plate directly to analysis), as well as the short analysis time (<2 min) before a determination can be made. These traits make MALDI-MS a powerful tool suited for environmental monitoring and identification of microbial hazards in drinking water.

PRESENTATION U.S. Environmental Protection Agency and Emerging Contaminants 05/13/2009
GLASSMEYER, S. U.S. Environmental Protection Agency and Emerging Contaminants. Presented at New Jersey institute of Technology Workshop, Newark, NJ, May 13, 2009.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-ug/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. To determine which wastewater chemicals persist through drinking water treatment, a follow-up study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 84 different emerging contaminants, including 24 pharmaceuticals. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-five chemicals were detected at least once, with 28 chemicals detected in the source waters and 23 chemicals detected in the finished waters. The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Surveillance Systems for Waterborne Protozoa: Beyond Method 1623 04/24/2009
VILLEGAS, E. Surveillance Systems for Waterborne Protozoa: Beyond Method 1623. Presented at Greater Cincinnati Water Works Science Advisory Board Meeting, Cincinnati, OH, April 24, 2009.
Abstract: 1. Brief introduction to waterborne Cryptosporidium and Giardia Historical perspective on detecting Cryptosporidium and Giardia Current detection methodologies 2. US EPA’s waterborne protozoan research program Building a “Protozoan Detection Toolbox” 3. Perspectives on the future of the “Protozoan Detection Toolbox” Future directions Factors to consider for developing a pathogen specific detection method

PRESENTATION Comparison of Enterococcus Qpcr Analysis Results from Fresh and Marine Water Samples on Two Real-Time Instruments 04/20/2009
HAUGLAND, R. A., M. VARMA, R. OSHIRO, J. Parr, AND M. Doolittle. Comparison of Enterococcus Qpcr Analysis Results from Fresh and Marine Water Samples on Two Real-Time Instruments. Presented at National Beaches Conference 2009, Huntington Beach, CA, April 20 - 22, 2009.
Abstract: EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the method’s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM and StepOnePlusTM series instruments that can retail for under $20 K and provide 48 or 96 sample analysis capacity. In this study we compared the results obtained on a StepOnePlusTM 96 well instrument with those obtained on the Cepheid Smart Cycler® which has been the primary source of the method’s results to date. Analyses were performed simultaneously on DNA extracts from multiple, replicate filter retentates of 12 marine and 12 freshwater samples from diverse locations using study and data analysis designs from EPA's microbial alternate test procedure protocol. Precision among log10 target sequence copy (TSC) estimates in the samples from the two instruments were compared with no significant difference (p > .05) based on the one-way ANOVA of Levene's Test for Homogeneity of Variance. Three–way ANOVA with fixed factors: instrument, matrix, instrument*matrix; and random factors: sample (nested in matrix) and inst*sample (nested in matrix) was used to compare the mean log10 TSC estimates with no significant difference seen between the instruments (p > .05). Given the wide variety of qPCR instruments that are already available and the likelihood that additional advances will occur in instrument technology, this study may provide a useful model for the design and implementation of additional comparative studies in the future.

PRESENTATION What Is the Relative Health Risk to Swimmers from California Seagull Feces Compared to Bather Shedders? 04/20/2009
Schoen, M. AND N. ASHBOLT. What Is the Relative Health Risk to Swimmers from California Seagull Feces Compared to Bather Shedders? Presented at National Beach Conference, Huntington Beach, CA, April 20 - 22, 2009.
Abstract: Estimated infection risks to swimmers from California seagull and bather sources of fecal contamination at a beach in Southern California were compared using quantitative microbial risk assessment (QMRA). The risk to swimmers of gastro-intestinal infections was estimated from Campylobacter jejuni, Cryptosporidium hominis, and Norovirus from human bathers and Campylobacter jejuni from Californian seagulls over the observed range of surfzone enterococci (ENT) concentration during normal summer conditions at Dohney Beach. A beta-Poisson dose-response model was utilized with pathogen specific parameters to calculate the probability of infection using Monte Carlo analysis of the uncertain input variables. Overall, the individual risks from C. jejuni and Norovirus were greater than that from C. hominis. Specific predictions of risk remain uncertain due to large uncertainty in model parameters; particularly the proportion of Campylobacter strains that are human infectious. If the proportion of infectious strains from gulls is low, near 0.01, then human infection risk from accidental ingestion of bathing water containing gull feces is only greater than the risk from bather shedders when gull fecal matter in the bathing waters exceeds that from humans by more than 20 times; that reduces to four times should the proportion of infectious C. jejuni gull strains be 0.05. The best estimate model results indicated that gull fecal-derived enterococci counts contribute less of a health threat to swimmers than human sources; however there remains large uncertainty in prediction due to the remaining uncertainty in human infectious campylobacter species in gull feces, their unknown environmental persistence and the level of bather shedding of human pathogens.

PRESENTATION Qpcr Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches 04/20/2009
CHERN, E. C., K. P. Brenner, L. J. WYMER, AND R. A. HAUGLAND. Qpcr Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches. Presented at EPA National Beaches Conference 2009, Huntington Beach, CA, April 20 - 22, 2009.
Abstract: The use of real-time qPCR to determine fecal indicator bacteria (FIB) densities is currently being investigated by the U.S. EPA. The present recreational water quality guidelines, based on culturable FIB, prevent same day determinations of water quality whereas results from the qPCR method can be available within several hours. Epidemiological studies at POTW-impacted freshwater beaches have shown a strong correlation between qPCR determined Enterococcus densities and swimming-related illness rates. This study provides an initial assessment of qPCR estimated Enterococcus, Bacteroidales, E. coli and Clostridium densities in marine water from two recreational beaches sampled over one summer. The estimated geometric mean cell densities per 100 ml of marine water from both beaches across sampling visits were 3.28 x 10 1, 1.71 x 103, 7.37 x 102, and 9.26 x 102 for Enterococcus, Bacteroidales, E. coli and Clostridium, respectively. These cell equivalent density estimates, determined using whole cell calibrator samples by a comparative cycle threshold (CT) approach, did not correspond with the relative target sequence density estimates of the different FIB in the samples which gave geometric means of 1.28 x 103, 2.35 x 104, 3.04 x 102, and 1.03 x 104 for Enterococcus, Bacteroidales, E. coli and Clostridium, respectively. This discrepancy was determined to be attributable to differences in recovery of target sequences from cells of the different organisms. QPCR analyses using whole cell calibrator samples provides a simple approach for comparing both total cell and target sequence density estimates of different FIB groups in water samples.

PRESENTATION Emerging Contaminants in the Drinking Water Cycle. 04/15/2009
GLASSMEYER, S. Emerging Contaminants in the Drinking Water Cycle. Presented at Region 3 Emerging Contaminants Webcast, The Web, Newark, NJ, April 15, 2009.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-g/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. To determine which wastewater chemicals persist through drinking water treatment, a follow-up study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 84 different emerging contaminants, including 24 pharmaceuticals. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-five chemicals were detected at least once, with 28 chemicals detected in the source waters and 23 chemicals detected in the finished waters. The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Postmodern Urban Infrastructure Pathogens and Other Scum Associates 03/11/2009
ASHBOLT, N. Postmodern Urban Infrastructure Pathogens and Other Scum Associates. Presented at Johns Hopkins, Washington, DC, March 11, 2009.
Abstract: To be presented at Johns Hopkins, Washington, DC, March 11, 2009

PRESENTATION An Investigation of Bioaccessibility of Arsenic in Rice Using Ic-ICP-MS 03/08/2009
Young, A. R., H. Trenary, S. K. Yathavakilla, P. A. CREED, J. T. CREED, AND J. XUE. An Investigation of Bioaccessibility of Arsenic in Rice Using Ic-ICP-MS. Presented at 2009 Pittcon , Chicago, IL, March 08 - 13, 2009.
Abstract: Arsenic exposure occurs mainly through drinking water and food; therefore, both aspects should be incorporated into any aggregate exposure assessment. Drinking water exposures are predominately inorganic arsenic while dietary exposures are made up of a diverse set of arsenicals with widely varying toxicities. Rice collected throughout the world has been shown to be relatively high in total arsenic. In fact, the FDA’s market basket survey supports this observation and for this reason rice is a target food group for dietary speciation studies. Arsenic speciation has been conducted on rice grown in endemic areas and in the U.S. In these studies, inorganic arsenic and DMA are the predominant arsenicals found in rice; however, the variation associated with the distribution of these two arsenicals is far from a constant. Therefore, a quantitative exposure assessment for rice needs to incorporate not only species specific information, but also the variation associated with the distribution of the arsenicals, in order to improve the exposure risk estimate for rice. Another factor which needs to be considered in the exposure assessment is the biologically relevant arsenic dose associated with rice consumption. Information on bioaccessible arsenic, the fraction of analyte which is solubilized by the gastrointestinal (GI) tract, could provide a means to estimating the biologically relevant dose. Ideally, this bioaccessibility term would also include the GI tract induced biotransformations associated with the ingested arsenicals. Currently, the species specific rice data are mainly comprised of chemically based extractions. These extractions are relatively simple and quantitative, but it is not known how accurately these extractions correlate with bioaccessibility. Therefore, an assay that can estimate the species specific bioaccessibility and capture the biotransformation in the GI tract should improve the exposure estimate. An application of this type of assay should provide data essential to estimating biologically relevant exposures in target foods. This presentation will attempt to estimate the bioaccessible fraction associated with U.S. consumed rice using a synthetic gastrointestinal extraction technique prior to speciation via ICICP-MS. Finally, the potential for biotransformation of the extracted arsenicals will be evaluated by using an in vitro technique in which a gastrointestinally extracted rice sample is incubated in the cecum content of a mouse.

PRESENTATION Rapid Methods for the Detection of General Fecal Indicators 02/11/2009
HAUGLAND, R. A. AND K. OSHIMA. Rapid Methods for the Detection of General Fecal Indicators. Presented at Gulf of Mexico Alliance, Microbial Source Tracking (MST) & Pathogens Detection Workshop, St. Petersburg, FL, February 11, 2009.
Abstract: Specified that EPA should develop: appropriate and effective indicators for improving detection in a timely manner of pathogens in coastal waters appropriate, accurate, expeditious and cost-effective methods for the timely detection of pathogens in coastal waters

PRESENTATION Surveillance Systems for Waterborne Cryptosporidium: US EPA Method 1523 and Beyond 02/08/2009
VILLEGAS, E. Surveillance Systems for Waterborne Cryptosporidium: US EPA Method 1523 and Beyond. Presented at 2009 USDA-CSREES National Water Conference, St. Louis, MO, February 08 - 12, 2009.
Abstract: Waterborne cryptosporidiosis remains a significant public health concern in countries around the world. Many species and genotypes of Cryptosporidium contaminate drinking water sources, but C. parvum and C. hominis remain the two predominant species known to cause waterborne disease outbreaks in humans. To improve human health and reduce risks posed by these pathogens, the US EPA promulgated the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule). This rule requires drinking water utilities to monitor for Cryptosporidium oocysts in their source waters using US EPA Method 1622 or 1623. These methods are designed to enumerate oocysts by microscopy and although very useful in determining concentration levels of Cryptosporidium oocysts in various drinking water sources throughout US, the methods are time consuming, labor intensive and cannot distinguish animal from human specific species or determine if the oocysts are infectious to humans. Because many Cryptosporidium spp. oocysts are morphologically similar and can contaminate drinking water supplies, the development of more specific detection and typing approaches for this parasite are essential to better understand the impact of this protozoan in source waters. The data generated by these methods will provide additional information that will be useful for future source water management strategies and for human health risk assessments related to Cryptosporidium oocyst contamination. Current research activities focused on developing new and more rapid molecular-based approaches (e.g., quantitative real-time PCR, microarrays, etc.) that can detect and determine the infectious potential of oocysts will be described. Advantages and inherent limitations of these approaches and their potential application(s) as alternative methods to current Cryptosporidium surveillance systems will be discussed.

PRESENTATION Can Qmra Be Used to Discount Pathogen Risk to Swimmers from Animal Fecal Contamination? Doheny Beach, Ca Case Study 12/08/2008
SCHOEN, M. E. AND N. ASHBOLT. Can Qmra Be Used to Discount Pathogen Risk to Swimmers from Animal Fecal Contamination? Doheny Beach, Ca Case Study. Presented at Society for Risk Analysis, Boston, MA, December 08 - 10, 2008.
Abstract: Estimated health risks to swimmers from seagull and bather sources of fecal contamination at Doheny Beach, California were compared using quantitative microbial risk assessment (QMRA) with a view to aiding beach closure decisions. Surfzone pathogens from seagulls were thought to be less of a human health threat than human fecal contamination. To evaluate if the seagull fecal contribution can be discounted, possibly reversing a beach closure, a discounting process is proposed here that estimated the risk to swimmers of gastro-intestinal illness and compared the predicted risk to the 19 illnesses per 1000 benchmark. The discounting process was exemplified for Campylobacter jejuni using 1) the counts of seagulls and swimmer/shedders and assumed pathogen load distributions, or 2) a hypothetical source tracking tool that measures the proportion of total fecal contamination attributed to the two sources. A beta-Poisson dose-response model was utilized to calculate the probability of infection using Monte Carlo analysis of the uncertain input variables. The conditions under which the seagull fecal contribution could be discounted were specified for the observed range of surfzone enterococci (ENT) concentration over the 2007 recreational season. However, even using a perfect source tracking tool, seagull fecal contributions could not be discounted for ENT concentrations above the 108 CFU/100ml single sample limit, at the 95% confidence level, given the 19/1000 benchmark. The same conclusion was drawn using fecal load estimates from the number of gulls, and various ratios to swimmers. The proposed discounting process negates the belief that gull fecal-derived enterococci counts contribute less of a health threat to swimmers than human sources, given the current large uncertainty in human infectious campylobacters in gull feces, let alone their unknown environmental persistence. The discounting process suggested could be applied to additional locations and sources, using appropriate reference pathogens for locations with different sources of fecal contamination.

PRESENTATION Viruses in U.S. Ground Waters: Hydrogeological and Methodological Data Gaps 12/02/2008
FOUT, G. Viruses in U.S. Ground Waters: Hydrogeological and Methodological Data Gaps. Presented at EPA Symposium on Groundwater-Borne Infectious Disease, Etiological Agents and Indicators , Washington, DC, December 02 - 04, 2008.
Abstract: Slide Presentation

PRESENTATION Reconfiguration of Water-Energy Systems for Healthy and Sustainable Urban Water Management 12/01/2008
ASHBOLT, N. Reconfiguration of Water-Energy Systems for Healthy and Sustainable Urban Water Management. Presented at International Conference on Water Scarcity, Global Changes, and Groundwater Management Responses, Irvine, CA, December 01 - 05, 2008.
Abstract: Presentation at the the International Conference on Water Scarcity, Global, Changes, and Groundwater Management Responses

PRESENTATION USEPA Approach for the Detection and Quantification of Enterococcus By Qpcr 12/01/2008
HAUGLAND, R. A. AND K. OSHIMA. USEPA Approach for the Detection and Quantification of Enterococcus By Qpcr. Presented at Standardization and International Evaluation of Molecular Methods Used for Detection of Waterborne Pathogens, Paris, FRANCE, December 01, 2008.
Abstract: The Beach Act 2000 specified that EPA should develop: Appropriate and effective indicators for improviding detection in a timely manner of pathogens in coastal waters Appropriate, accurate, expeditious and cost-effective methods for the timely detection of pathogens in coastal waters.

PRESENTATION EPA Research and Development: National Exposure Research Laboratory 11/21/2008
PAWLECKI-VONDERHEIDE, A. M. EPA Research and Development: National Exposure Research Laboratory. Presented at University of Cincinnati, Department of Biological Science, Cincinnati, OK, November 21, 2008.
Abstract: This course is for Biology majors, primarily those in the completed Freshman Biology. Students enrolled in the course are expected to have completed Freshman Biology. With some background in biology as a strt, students begin to think about doing some research as part of their undergraduate education, but they frequently do not have much of an idea of what is involved. This course will serve as an introduction to different types of research in biology. Studnets will have the opportunity to interact with individuals who are carrying our active research programs in a variety of different settings. Students will be introduced to the primary literature by reading articles published by the presenters. With presenters from different research environments, students will be able to learn about different careers in biology.

PRESENTATION Real-Time Qpcr Data Analysis and Determination of Method Performance 11/16/2008
HAUGLAND, R. A. Real-Time Qpcr Data Analysis and Determination of Method Performance. Presented at WQTC Conference Workshop, Cincinnati, OH, November 12 - 16, 2008.
Abstract: Quantitative PCR for the Water Mmicrobiologist course material to be presented at the WQTC Conference Workshop, Cincinnati, OH, November 16, 2008

PRESENTATION Environmental Applications of Qpcr 11/16/2008
PFALLER, S. L. Environmental Applications of Qpcr. Presented at 2008 Water Quality and Technology Conference Workshop, Cincinnati, OH, November 16 - 20, 2008.
Abstract: Slide presentation given at the WQTC Workshop titled: Quantitative PCR for the Water Microbiologist: An Introductory Course

PRESENTATION Cryptosporidium Off-the-Slide Genotyping: Recovery of Oocysts Extracted from Slides Examined By Method 1623 11/16/2008
WARE, M. W. AND E. VILLEGAS. Cryptosporidium Off-the-Slide Genotyping: Recovery of Oocysts Extracted from Slides Examined By Method 1623. Presented at Water Quality Technology Conference, Cincinnati, OH, November 16 - 20, 2008.
Abstract: Cryptosporidium has been found world wide and is an important waterborne parasite causing a self-limiting diarrhea; however, in the immunocompromised it may become chronic and can even lead to death. To characterize the risk of exposure to Cryptosporidium in water the USEPA enacted Long Term 2 Enhanced Surface Water Treatment Rule (LT2) requiring United States surface water utilities to monitor for Cryptosporidium using USEPA Method 1623 (4, 5). This microscopic microscopy based method enumerates oocysts in a water sample,; but it can not determine oocyst species or identify the strain. Cryptosporidium speciation and genotyping can be achieved by molecular methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Studies of environmental water and wild life fecal samples reveal that several isolates and species from wildlife and livestock animals are more frequently identified than the two principle human pathogens C. hominis and C. parvum (6-8). Several approaches in the literature and as well as one commercial product have been developed to genotype oocysts detected on the well slide wells following examination ed by Method 1623 (1-3). These genotyping methods require extracting oocysts from the slide by removing the coverslip, scrapping the well to detach the oocysts, and transferring the oocysts to a tube for for molecular analysis. One of the procedures also includes the removales of the DABCO containing mounting media, which could inhibitmay inhibit PCR. These approaches have not been compared and validated in an independent study. If one or all of these approaches are robust enough to in which the oocysts are recovered efficiently recover oocysts, then this “off-the-slide” genotyping would greatly provide additional genotypinformation ing results tohat would improve the quality of the monitoring data gathered for assessing risk. However, if this is not the caseOn the other hand, the genotyping results may may have the potential to create false negatives by not detecting all of the species present or may indicate a false positive by Method 1623, creating data interpretation problems. This study focuses on recovery efficiencies of whole C. parvum oocysts using “off-the-slide” extraction techniques. The extraction approach was evaluated in two phases: 1) removal of the coverslip and mounting media, and 2) transfer of the oocysts from the slide to the genotyping tube. C. parvum oocysts were counted by flow cytometery to contain 200, 20 or 0 oocysts per well. The oocysts were either pre-stained or stained on the well. The and slides were counted by microscopy. then tThe coverslip was then removed and retained, and the well was washed. The coverslip and washed well were recounted. Results show that the number of oocysts remaining on the well was quite variable after removal of the coverslip and mounting media, ranging from nearly 5095% too nearly 9550%. Less than 2% of the oocysts on average were attached to the coverslip, with the remaining oocysts removed with the mounting media. These results show suggest that removal of the coverslip accounts for little oocysts loss; however, the mounting media retained on the slide must be transferred to the genotyping tube to accurately reflect the oocysts on contents of the slide. Phase 2 evaluated the transfer of oocysts from the slide to the genotyping tube. The methods used were similar to those used in phase 1 except that the surface of the well was scraped three times with a 10 l loop, as previously described prior to transfer (1-32) and the oocysts were transferred through a 0.8 m filter which was then restained and counted. The scrapped well and coverslip were also examined and only a few oocysts were observed on the scrapped well and coverslip, indicating the scrapping efficiently removes oocysts attached to the well slide. The overall oocyst average transfer rate and its implications for the utility of off-the-slide genotyping will be discussed.

PRESENTATION Wqtc 2008 Workshop Quantitative Pcr for the Water Microbiologist: An Introductory Course 11/16/2008
BRINKMAN, N. Wqtc 2008 Workshop Quantitative Pcr for the Water Microbiologist: An Introductory Course. Presented at American Water Works Association's Water Quality and Technology Conference, Cincinnati, OH, November 16 - 20, 2008.
Abstract: Course presentation to be given at the American Water Work's Association's Water Quality and Technology Conference, Cincinnati, OH, November 16-20, 2008

PRESENTATION Qpcr Analysis of Total and Viable Enterococci in Waste Water Treatment: Comparison With Culture in Predicting Pathogen Reductions 11/16/2008
VARMA, M., R. A. HAUGLAND, R. Field, M. K. STINSON, B. Rukovets, AND L. J. WYMER. Qpcr Analysis of Total and Viable Enterococci in Waste Water Treatment: Comparison With Culture in Predicting Pathogen Reductions. Presented at Water Quality Technology Conference, Cincinnati, OH, November 16 - 20, 2008.
Abstract: BEACH Act amendment to Clean Water Act requires EPA to establish more expeditious methods for the timely detection of pathogens and pathogen indicators in coastal waters New methods should demonstrate utility for and be compatible with all CWA 304(a) criteria needs including: Water quality assessment for public notification at beaches Assessment of impaired waters listings Development of total daily maximum loads (TMDL) Development of National Pollution Discharge Elimination System (NPDES) permits Considerable progress has been made in the development of a qPCR method for monitoring DNA target sequence concentrations of enterococci fecal indicator bacteria that meets the BEACH act and CWA 304(a) requirements for water quality assessment at beaches

PRESENTATION USEPA-Usgs Case Studies 11/16/2008
GLASSMEYER, S. USEPA-Usgs Case Studies. Presented at Society of Environmental Toxicology and Chemistry Annual Meeting, Tampa, FL, November 16, 2008.
Abstract: The presence of emerging contaminants (ECs) in the environment is an area of growing concern for the scientific community and the general public. ECs include a broad range of chemicals including personal care products, pharmaceuticles, industrial compounds, pesticides, and hormones. A large body of evidence indicates that ECs are widespread in aquatic and terrestrial systems and have the potential to affect the health of organisms. ECs can affect development, metabolism, growth, osmoregulation, reproduction, responses to stress, and immune functions. Despite their potential ecosystem impact, their environmental fate and effects are still poorly understood in many regards. To better study and understand these potential impacts chemistry and toxicology sciences need to be linked. This short-course will emphasize what is currently known about the environmental fate and effects of ECs and the potential implications for populations and ecosystems, Select chemical and biological tools developed and improved for this research will be discussed as well as highlighting the challenges linking environmental exposure to ECs to effects.

PRESENTATION Development of a U.S. EPA Method for the Analysis of Selected Drinking Water Contaminants By LC/MS/MS 11/16/2008
SHOEMAKER, J. A. AND B. BOUTIN. Development of a U.S. EPA Method for the Analysis of Selected Drinking Water Contaminants By LC/MS/MS. Presented at Water Quality Technology Conference, Cincinnati, OH, November 16 - 20, 2008.
Abstract: The U.S. Environmental Protection Agency’s (U.S. EPA) Office of Ground Water and Drinking Water (OGWDW) collects national occurrence data on drinking water contaminants using Unregulated Contaminant Monitoring Regulations (UCMRs). These contaminants may be selected from the Drinking Water Contaminant Candidate List (CCL), or may be emerging contaminants with the potential for inclusion on future CCLs. In order for a contaminant to be included in a future UCMR, a standardized analytical method for its measurement in drinking water must be available. A group of 13 chemical contaminants are being evaluated for inclusion in a direct aqueous injection-liquid chromatography/tandem mass spectrometry (DAI-LC/MS/MS) method. Preliminary data were obtained for all 13 chemical contaminants. For chlorinated tap water fortified at 10 ug/L, recoveries ranged from 83 to 106% with a relative standard deviation of 0.8-7.9% for 11 of the 13 chemicals.

PRESENTATION Emerging Contaminants in US Drinking Water: Do You Know Where Your Water's Been? 11/16/2008
GLASSMEYER, S., E. Furlong, D. Kolpin, R. J. MILTNER, AND S. Werner. Emerging Contaminants in US Drinking Water: Do You Know Where Your Water's Been? Presented at Society of Environmental Toxicology and Chemistry Annual Meetingq, Tampa, GA, November 16, 2008.
Abstract: The drinking water and wastewater cycles are integrally linked. Chemicals that are present in household wastewater may be sufficiently mobile and persistent to survive on-site or municipal wastewater treatment and post-discharge environmental processes. Such compounds have the potential to reach surface and ground waters. These downstream / down gradient waters are typically the source for another community’s drinking water. To determine which wastewater chemicals persist through drinking water treatment, a joint USEPA / USGS study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 96 different emerging contaminants, including 35 pharmaceuticals, at sub-ug/L levels. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-nine chemicals were detected at least once, with 32 chemicals detected in the source waters and 26 chemicals detected in the finished waters. Overall, the most frequently detected chemicals were aspirin (60 %), bupropion (60 %), caffeine (40 %), carbamazepine (40 %), tri(2-chloroethyl) phosphate (40 %) and venlafaxine (40 %). The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In general, the results from locations that used conventional treatment (coagulation, clarification, filtration, and chlorination) showed negligible removal of the monitored emerging contaminants. The results from those sites that used more advanced treatment (granular activated carbon adsorption, ozonation or UV irradiation) showed greater removal percentages.

PRESENTATION Research Underpinning New EPA Rec Water Quality Criteria: Quantitative Microbial Risk Assessment and Predictive Models 10/30/2008
ASHBOLT, N. Research Underpinning New EPA Rec Water Quality Criteria: Quantitative Microbial Risk Assessment and Predictive Models. Presented at Water Quality Conference: How Clean or Polluted are Hawaii's Waters, Honolulu, HI, October 30 - 31, 2008.
Abstract: Slide presentation to be given at the Water Quality Conference: How Clean or Polluted are Hawaii's Waters, October 30-31, 2008, Honolulu, Hawaii

PRESENTATION Sustainable Urban Waterfutures: A Vision 10/16/2008
ASHBOLT, N. Sustainable Urban Waterfutures: A Vision. Presented at Federal Subcommittee on Water Availability and Quality Executive Office of the President, Washington, DC, October 16, 2008.
Abstract: Background: Urban growth is seriously limited by water scarcity on every continent, and trying to house more people that aspire to current developed region water services is simply impossible due to lack of available water, let alone the cost. Furthermore, traditional water/sanitation services are net users of energy (e.g. 10% of US energy production is used in drinking and wastewater services), whereas the biodegradable organic matter within wastewater contains over nine times as much energy as typically used to treat wastewater in developed regions. However, an often overlook third key point here is the finite nature of agriculturally-required phosphorus (P, mined from rock phosphate reserves) that is expected to be fully exploited in 60-150 years time (variability depending on global food/biofuel production increases). Hence, it should be obvious that ‘wastewater’ nutrients, and in particular P need to be recycled for agriculture to be sustainable, not to mention the energy savings that would result from co-recycling nitrogen and potassium. Why do we have unsustainable water services? Despite our centralized water services being engineered to meet public health protection, there has been a series of misinformed decisions that provide us with today’s legacy – starting in the mid 1800’s when London’s engineer Sir Joseph Bazalgette instituted major sewers systems on the basis that bad air (miasma) resulting from human wastes fouling the Thames River were the causes of cholera and typhoid. The word Sewer means "seaward" in Old English, and that was the start of the ‘solution to pollution was dilution’ instituted by Queen Victoria and used by many sanitary engineers to this day. The large-scale introduction of the flushing toilet from the 1890’s only exacerbated the need for larger sewers and waterworks, with the latter primarily articulated throughout cities for fire fighting (=reduced house insurance premiums), with only some 10% required for drinking water purposes. Also lost was the concept of ‘night soil’ for nutrient recycling to agriculture. A more sustainable path for water services: Using the sustainability principle of source-separation of waste streams, there is a range of options with household sanitary streams for nutrient and energy recovery (Figure 1). However, such changes require different incentives and institutional structures/relationships to flourish. For example, peoples’ recent enthusiasm for hybrid/electric cars is sadly not for altruistic reasons, but simply due to the sudden increase in fuel costs. In many situations, more decentralized water systems (possibly still centrally managed) may not only provide the means for local energy recovery ‘stations’, but also household greywater reuse that would reduce drinking water demand by over 70% and reduce the need for large water mains, sewers and pumping stations. Hence, incentives need to be abundant to aid in this translation to more sustainable urban water systems. Experience from projects in Sweden and Australia that are based on participatory, iterative processes of agreement for more sustainable systems will be discussed.

PRESENTATION Influence of Activity on Transfer of Pesticides from Treated Formica ® in Foods 10/12/2008
MELNYK, L. J., T. E. HIEBER, T. Turbeville, J. N. MORGAN, AND A. M. PAWLECKI-VONDERHEIDE. Influence of Activity on Transfer of Pesticides from Treated Formica ® in Foods. Presented at International Society of Exposure Analysis, Pasenda, CA, October 12 - 16, 2008.
Abstract: The children’s dietary intake model (CDIM) has been developed to predict total dietary intake of a child incorporating excess exposures due to handling of food prior to consumption (Akland et al., 2000; Melnyk et al., unpublished). The model includes three Terms added together to estimate intake of a single contaminant. Term 1 is the pesticide residue in the food; Term 2 is the contamination due to surface-to-food contacts; and Term 3 is surface-to-hand-to-food contamination. The data for Term 1 are obtained through duplicate diet analysis or generated from residue and consumption information from available databases. The second and third input parameters may be generated from laboratory and field studies and the measurement procedure involves contaminant transfers to foods from surfaces and the frequency and duration of activities resulting in the contacts. Previous studies, conducted in the laboratory, determined transfer efficiencies between certain food and surface combinations (Rohrer et al., 2003; Vonderheide et al., 2008). The impact of activity on the transfer of contaminants to foods is largely unknown or has been estimated based on multiple touches of a single food item (Melnyk et al., unpublished). Additional laboratory studies to determine the influence of activities on the transfer of contaminants to foods were performed in simulation experiments in which food was exposed to treated Formica® under specific conditions. The objectives of the experiments were to measure the impact of the activities to properly utilize the activity factor within the CDIM. This information will allow for better predictions by CDIM for children’s total dietary exposure which, in turn, will lead to more accurate assessments for risk analysis.

PRESENTATION Apples: A Standard Food for Determining Potential Residential Pesticide Transfers 10/12/2008
MELNYK, L. J., C. Stewart, T. E. HIEBER, J. N. MORGAN, AND A. M. PAWLECKI-VONDERHEIDE. Apples: A Standard Food for Determining Potential Residential Pesticide Transfers. Presented at International Society of Exposure Analysis, Pasenda, CA, October 12 - 16, 2008.
Abstract: Children’s unstructured eating behaviors lend themselves to potential dietary exposures to synthetic pyrethroid pesticides applied in the home. To determine the potential for excess dietary exposure of children from handling food during consumption, a standard food has been developed to be used in the field as a measure of transfers of pesticides. The standard food of choice was a raw Red Delicious apple because of its consistent transfer efficiencies for a range of pesticides and its ease of analysis. Since this type of apple may not be available everywhere, various types of apples were studied to determine if the type impacted the transfer of pesticides from contaminated Formica®. Also, in the field, preparation of the apple slices could vary. The cutting technique and time lapse between cut and use of the slice of raw apple were investigated for their impact on the transfer of pesticides from a surface. The goal was to develop a standard operating procedure to be used in the field for residential monitoring of potential excess dietary exposures of children.

PRESENTATION Interferon Gamma as a Biomarker of Exposure to Enteric Viruses 10/12/2008
LI, L. AND A. P. DUFOUR. Interferon Gamma as a Biomarker of Exposure to Enteric Viruses. Presented at 2008 Joint ISEE-ISEA Conference, Pasenda, CA, October 12 - 16, 2008.
Abstract: Interferon gamma (IFN-γ) was selected as a biomarker for viral exposure. Twelve-week-old BALB/c mice were intraperitoneally injected with Coxsackievirus B3 or B4 diluted in phosphate-buffered saline (PBS). Control mice were injected with PBS only. Four months after viral infection, T lymphocytes were isolated from mouse thymus and spleen. T lymphocyte release of IFN-γ was examined after in vitro incubation with viral antigens, a mitogen, PHA and PBS respectively. The level of IFN-γ released by T lymphocytes was examined by antibody-capture chemiluminescent ELISA. A marked increase in level of IFN-γ was observed when T cells from Coxsackievirus B3-infected mice were incubated with B3 virus but not B4 or PBS. This indicated that Coxsackievirus B3- sensitized T cell receptor recognized only T cell epitope from B3 virus not B4. Coxsackievirus B4 primed mouse T cells did not release IFN-γ when they were incubated with B3 virus or PBS. Our results showed that IFN-γ produced by primed memory T cells is virus-specific. The data also show that memory T cells can distinguish very structurally related Coxsackievirus B3 and B4 when the sensitized T cells were directly stimulated by the viruses in vitro. The results of this study may be extended to human exposure studies related to microbial pathogens.

PRESENTATION Emerging Contaminants in the Drinking Water Cycle 10/07/2008
GLASSMEYER, S. Emerging Contaminants in the Drinking Water Cycle. Presented at AWWA Alabama-Mississippi Regional Meeting, Montgomery, AL, October 06 - 07, 2008.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-µg/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50% wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. In the source water of one conventional drinking water facility, 45 out of 113 monitored chemicals were detected at least once, with 21 chemicals still detectable in the finished drinking water. This documents the incomplete removal for some chemicals during treatment. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Emerging Contaminants in US Drinking Water: Do You Know Where Your Water's Been? 10/03/2008
GLASSMEYER, S., E. Furlong, D. Kolpin, R. J. MILTNER, AND S. Werner. Emerging Contaminants in US Drinking Water: Do You Know Where Your Water's Been? Presented at Society of Environmental Toxicology and Chemistry Ohio Valley Chapter Annual Meeting, Bloomington, IN, October 03, 2008.
Abstract: The drinking water and wastewater cycles are integrally linked. Chemicals that are present in household wastewater may be sufficiently mobile and persistent to survive on-site or municipal wastewater treatment and post-discharge environmental processes. Such compounds have the potential to reach surface and ground waters. These downstream / down gradient waters are typically the source for another community’s drinking water. To determine which wastewater chemicals persist through drinking water treatment, a joint USEPA / USGS study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 96 different emerging contaminants, including 35 pharmaceuticals, at sub-ug/L levels. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-nine chemicals were detected at least once, with 32 chemicals detected in the source waters and 26 chemicals detected in the finished waters. Overall, the most frequently detected chemicals were aspirin (60 %), bupropion (60 %), caffeine (40 %), carbamazepine (40 %), tri(2-chloroethyl) phosphate (40 %) and venlafaxine (40 %). The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In general, the results from locations that used conventional treatment (coagulation, clarification, filtration, and chlorination) showed negligible removal of the monitored emerging contaminants. The results from those sites that used more advanced treatment (granular activated carbon adsorption, ozonation or UV irradiation) showed greater removal percentages.

PRESENTATION Analysis of Arsenicals and Their Sulfur Analogs in Biological Samples Using Hplc With Collision Cell ICP-MS and Esi-MS/MS 09/28/2008
GALLAWA, C., K. KUBACHKA, P. A. CREED, J. T. CREED, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Analysis of Arsenicals and Their Sulfur Analogs in Biological Samples Using Hplc With Collision Cell ICP-MS and Esi-MS/MS. Presented at 35th FACSS, Reno, NV, September 28 - October 02, 2008.
Abstract: Recent arsenic speciation studies have indicated that the sulfur analogs of the more common arsenic oxides are present in environmental and biological systems. This discovery was previously impeded due to the strong affinity of these arsenic-sulfides for the stationary phases typically used in arsenic oxide based speciation studies. The presence of thiolated arsenicals adds to the chromatographic resolution requirements essential for elemental-based detection (ICP-MS), mainly because misidentifications of an unknown can be made when relying solely on retention time matching when using element specific detection systems. The need for complementary molecular based information using techniques, like LC-ESI-MS/MS, have proven beneficial for assigning molecular structures to unknown arsenicals within complex matrices. The need is also especially important in indentifying the thiolated arsenicals, because primary standards of the neat materials are not commercially available. The detection of thiolated arsenicals as urinary metabolites in both animals and humans has raised questions regarding how and when these species are produced within the metabolic pathway of arsenic. The authors have previously indicated that the microflora in the cecum of a mouse are able to biotransform an arsenosugar oxide (a tri-alkyl substituted arsenic oxide) to its corresponding sulfide [1]. This biotransformation raises questions regarding the conversion of structurally similar arsenic oxides, such as dimethylarsinic acid (DMA, di-alkyl substituted arsenic oxide). This presentation will summarize the analytical data from both LC-ICP-MS and LC-ESI-MS/MS that confirms the production of dimethylthioarsinic acid (DMTA) in the cecum of a mouse. The presentation will also report on the use of an isotopically enriched sulfur label to distinguish between proposed metabolic pathways. Finally, some preliminary research on the conversion of inorganic arsenic to its sulfur analogs will be presented.

PRESENTATION Verification of Cryptospporidium Removal By a Full-Scale Water Treatment Plant Using a Yeast Challenge and Ramifications for Risk Assessments 09/26/2008
ASHBOLT, N. Verification of Cryptospporidium Removal By a Full-Scale Water Treatment Plant Using a Yeast Challenge and Ramifications for Risk Assessments. Presented at The Greater Cincinnati Water Works Advisory Committee, Cincinnati, OH, September 26, 2008.
Abstract: Presentation at the Greater Cincinnati Water Works Advisory Commmittee, Cincinnati, September 26, 2008

PRESENTATION Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time Qpcr Analysis of Enterococci in Recreational Waters 09/15/2008
SIEFRING, S., R. A. HAUGLAND, AND L. J. WYMER. Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time Qpcr Analysis of Enterococci in Recreational Waters. Presented at Great Lakes Beaches Association Annual Meeting, Porter, IN, September 15 - 17, 2008.
Abstract: In response to the Beach Act, the U.S. EPA has developed a quantitative PCR (qPCR) method for enterococci that meets requirements for rapid, risk-based water quality assessments of recreational waters. Widespread implementation of this method will require that different laboratories obtain consistent quantitative results. An important prerequisite will be the availability of reproducible, stable and sustainable standards. In this study two commercial sources of enumerated and lyophilized Enterococcus cells, BioBallTM (BTF, Ltd.) and EpowerTM (MicroBioLogics, Inc.) were used to prepare calibration samples for the qPCR method. The precision and recovery of target sequences from multiple samples prepared from each these cell sources and from currently used laboratory cultured cells were determined from qPCR cycle threshold (CT) results and by comparisons with a standard curve from known target sequence quantities. Unlike cultured cells, samples prepared from both of these products required filtration to remove PCR-inhibitory materials before DNA extraction. The ratios of variances in target sequence recoveries from replicate Epower and BioBallTM samples were 2.48 (p = 0.068) and 1.10 (p = 0.433), respectively, compared to the cultured cell samples. Lower precision obtained from the BioBallTM samples may be related to the lower cell quantities in these samples (~550 CFU vs. ~10^5 CFU in the EpowerTM and cultured cell samples). Absolute recoveries of target sequences per CFU from the three cell sources were similar. Each of these products has potential advantages in either cost (EpowerTM) or ease of use and more appropriate cell numbers for quantifying recreational water sample results (BioBallTM).

PRESENTATION International Use of Risk Assessment/Risk Management to Direct Waterborne Pathogen Research 09/04/2008
ASHBOLT, N. International Use of Risk Assessment/Risk Management to Direct Waterborne Pathogen Research. Presented at Pathogens-In-Groundwater Research Consortium National Workshop, Calgary, AB, CANADA, September 04 - 05, 2008.
Abstract: Presentation to given at the Pathogens-In-Groundwater Research Consortium National Workshop, Delta Bow Valley, Calgary, Canada, September 4-5, 2008

PRESENTATION From Source-to-Tap; Understanding the Ecology of Environmental Pathogens for Sustainable Urban Water Use 08/20/2008
ASHBOLT, N. From Source-to-Tap; Understanding the Ecology of Environmental Pathogens for Sustainable Urban Water Use. Presented at The Water Cycle ISME, Cairns, AUSTRALIA, August 20, 2008.
Abstract: Presentation given at The Water Cycle ISME, Cairns, Australia, August 20, 2008

PRESENTATION Visible Light-Activated Tio2 Photocatalytic Films; Synthesis, Characterization and Environmental Application for the Destruction of Microcystin-Lr 08/17/2008
Pelaez, M. A., A. A. DELACRUZ, AND D. D. Dionysiou. Visible Light-Activated Tio2 Photocatalytic Films; Synthesis, Characterization and Environmental Application for the Destruction of Microcystin-Lr. Presented at 236th American Chemical Society National Meeting, Philadelphia, PA, August 17 - 20, 2008.
Abstract: Titanium dioxide (TiO2) photocatalysis has become one of the most effective advanced oxidation technologies (AOTs) for the treatment of persistent organic contaminants. To generate hydroxyl radicals, a non-selective, reactive oxidizing species and responsible for the oxidation of pollutants, conventional TiO2 must be activated under UV light irradiation (<400nm). This represents a limitation for the use of sustainable technologies with renewable energy sources such as solar light, where only 5% of the total spectrum includes UV irradiance. Several techniques have been established and demonstrated some success to doped TiO2 with dye, metals and non-metals to shift the absorption spectrum towards the visible light [1, 2]. More recent studies dealing with anionic-doped TiO2 (i.e nitrogen-doped TiO2) catalyst have reported an improvement in the photocatalytic activity in the visible region (<500 nm) [3, 4]. A particular technique, sol-gel method, has shown promising results for the formation of nitrogen doped TiO2 from environmentally friendly molecular precursors via room temperature procedures [4]. However, most of the studies so far on the nitrogen doped TiO2 materials were not directed towards its application in engineered water treatment processes. For instance, an emerging group of contaminants of primary concern worldwide are the cyanobacteria toxins. These are released from cyanobacterial harmful algal blooms (Cyano-HABs) and are considered a serious health risk due to their high solubility in water, toxicity (i.e., hepatotoxicity, neurotoxicity) and chemical stability [5]. Microcystin-LR (MC-LR) is one of the most commonly detected cyanotoxin in Cyano-HABs and the most toxic derivative of the group of microcystins [6]. High degradation rates of MC-LR were recently obtained with nitrogen-doped TiO2 nanoparticles synthesized by sol-gel based methods [4]. An implication of using suspended TiO2 nanoparticles is their possible mobility and transport in the environment, representing a health risk and requiring an additional filtration step for removal in processed water. This study investigates immobilized TiO2 catalyst with visible light sensitization with enhanced structural properties using a novel sol-gel process. Results on the evaluation of the films on the destruction of MC-LR will be presented.

PRESENTATION Development of a Gas Chromatography/Mass Spectrometry Method for the Analysis of the Solvent Stabilizer 1,4-Dioxane in Drinking Water 08/11/2008
GRIMMETT, P. AND J. W. MUNCH. Development of a Gas Chromatography/Mass Spectrometry Method for the Analysis of the Solvent Stabilizer 1,4-Dioxane in Drinking Water. Presented at 24th Annual National Environmental Monitoring Conference, Washington, DC, August 11 - 15, 2008.
Abstract: The solvent stabilizer 1,4-dioxane was named to the latest draft Drinking Water Contaminant Candidate List (CCL3) in February 2008 by the United States Environmental Protection Agency (USEPA). To collect occurrence data under the Unregulated Contaminant Monitoring Regulation (UCMR) program, a standardized method that exhibits ruggedness, accuracy, and precision is needed. Analysis of 1,4-dioxane has proved challenging because its volatility and miscibility with water make the compound a poor candidate for traditional extraction and concentration techniques. USEPA’s National Exposure Research Laboratory (NERL) has developed a new method, employing an activated carbon solid phase extraction, with quantitation performed by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode. Using the method parameters, 1,4-dioxane (1.0 µg/L) was recovered from groundwater, surface water, and surface water high in total organic carbon (TOC) at efficiencies of 96%, 99%, and 102%, respectively, using 500-mL drinking water samples. Relative standard deviations (RSD) were less than 6% for all drinking water sources (n = 7). Small-scale extractions using 100-mL water samples yielded comparable results. Method detection limits (MDL) calculated from fortified water samples analyzed at three laboratories were 0.012 µg/L, 0.020 µg/L, and 0.021 µg/L, with lowest concentra¬tion minimum reporting level (LCMRL) values of 0.013 µg/L, 0.036 µg/L, and 0.080 µg/L. Drinking water samples preserved with sodium bisulfate, dechlorinated with sodium sulfite, and stored under refrigeration were stable for 28 days.

PRESENTATION Legionella - (re-) awakening to the amoeba-based pathogens of distribution system biofilms 07/24/2008
ASHBOLT, N. Legionella - (re-) awakening to the amoeba-based pathogens of distribution system biofilms. Presented at With Environmental Research Laboratory, Tucson, AZ, July 24, 2008.
Abstract: Presentation to be given at the Environmental Research Laboratory, University of Arizona, Tucson, Arizona, July 24, 2008

PRESENTATION Legionella - (re-)awakening to the Amoeba-based Pathogens of Distribution System Biofilm 07/15/2008
ASHBOLT, N. Legionella - (re-)awakening to the Amoeba-based Pathogens of Distribution System Biofilm. Presented at Center for Biofilm Engineering, Bozeman, MT, July 15, 2008.
Abstract: Fecal pathogens have long been the focus of concern in the distribution of drinking waters. Yet today, with distribution system ‘failures’ accounting for the majority of waterborne outbreaks in the USA, there is growing realization that pathogens endemic to aquatic biofilms may also be of concern, particularly to susceptible sub-populations. These concerns have focused on strains of Legionella spp., Mycobacterium avium complex and Helicobacter pylori, as raised in EPA’s contaminant candidate lists (CCL-1, 2 & 3). Such pathogens, however, may only be the tip of the ‘iceberg’. Turning to biofilm ecology, it is clear from studies on Legionella that biofilm amoeba play an important ‘Trojan Horse’ role by introducing, amplifying and possibly up-regulating virulence in Legionella and other pathogens. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization, parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles into drinking water distribution systems. Moreover, the implications of these environmental niches for drinking and reuse water exposures to pathogenic legionellae are poorly understood. On-going research at EPA’s Cincinnati laboratories into the putative role of biofilms and amoeba in the proliferation, development and dissemination of potentially pathogenic Legionella and other intracellular pathogens will be discussed. The goal of this research is to aid in identifying control strategies and to provide key data to enable quantitative microbial risk assessments of biofilm-associated pathogens.

PRESENTATION Loss of Virus-Specific Memory T. Cells in Coxsackievirus B3 and B4 Infected Mice 06/05/2008
LI, L. AND A. P. DUFOUR. Loss of Virus-Specific Memory T. Cells in Coxsackievirus B3 and B4 Infected Mice. Presented at FOCIS Meeting, Boston, MA, June 05 - 09, 2008.
Abstract: There are two major types of enteroviruses: polioviruses and non-polio enteroviruses. While vaccines have effectively eliminated poliovirus infections, no vaccine is currently available for the non-polio enteroviruses. Generation of long-term pathogen specific memory cells is critical for the development of a good vaccine. To investigate whether long-term memory T cells are produced in response to infection by non-polio enteroviruses, coxsackieviruses B3 (CVB3) and coxsackieviruse B4 (CVB4) were intraperitoneally inoculated into adult BABL/c mice. Virus-specific memory T cells were assayed for proliferation and release interferon gamma (IFN-γ) under ex vivo stimulation with the viral pathogens. The level of IFN-γ was measured by antibody-capture chemiluminescent ELISA. Four days after stimulation, there were no detectable changes in the levels of IFN-γ from mice 17-19 months post-exposure to CVB3 and CVB4 compared with negative control mice inoculated only with sterilized PBS buffer. In contrast, the levels of IFN-γ were markedly increased after stimulation with specific viral pathogens in mice 2½ months post-infection with CVB3 and CVB4. These results suggest that non-polio enteroviruses might generate only short-lived virus-specific memory T cells. This would imply that vaccines for non-polio enterviruses might not provide long term protection against these viruses.

PRESENTATION Detection of Mycobacterium Avium Subsp. Paratuberculosis in Drinking Water and Biofilms Using Quantitative Pcr 06/05/2008
Beumer, A., D. N. KING, AND S. L. PFALLER. Detection of Mycobacterium Avium Subsp. Paratuberculosis in Drinking Water and Biofilms Using Quantitative Pcr. Presented at American Society for Microbiology General Meeting, Boston, MA, June 01 - 05, 2008.
Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. Cows infected with Johne’s disease shed large quantities of MAP into soil. Further, MAP has been isolated from surface water, is resistant to chlorine and able to form biofilms; therefore, it is likely to colonize drinking water distribution systems and be found in drinking water. Occurrence of MAP in drinking water has never been demonstrated. Because MAP is extremely difficult to isolate from water quantitative PCR (QPCR) assays have been developed to identify the presence and estimate the number of MAP. We used primer and probe sets designed to target IS900 sequences in the MAP genome, and we confirmed our IS900 results using Target 251 which is known to be specific to MAP. Drinking water was collected from 31 cold water faucets in southwestern Ohio. Two one liter samples were collected: a first pull sample, taken when the water was first turned on, and a standard method sample taken after running the water two minutes. The first pull is intended to collect cells from household pipes and potentially cells sloughed from biofilms; the standard method sample is intended to collect cells primarily from the water column. Biofilms were collected by swabbing the entire faucet grating, followed by vortexing in sterile water to remove attached biofilm. Water and biofilm samples were filtered, DNA was extracted and analyzed using QPCR (IS900 and Target 251 primers). Eighty four percent of first pull samples were positive for MAP, 92% of standard methods samples were positive, and 89% of biofilm samples were positive for MAP. Numbers of MAP (assuming 18 copies IS900 per organism) ranged from 0 to 29,000; however 82% of samples had less than 500 organisms per liter or faucet biofilm. There does not appear to be any statistical difference between presence in the drinking water distribution system and biofilm or first pull samples. This study shows that MAP is common in drinking water distribution systems, residential pipes and residential biofilms.

PRESENTATION Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary By Real-Time Quantitative Pcr 06/01/2008
CHERN, E. C., M. VARMA, M. Byappanahali, R. Whitman, R. Zepp, AND R. A. HAUGLAND. Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary By Real-Time Quantitative Pcr. Presented at 108th General Meeting of the American Society for Microbiology, Boston, MA, June 01 - 05, 2008.
Abstract: The Salt Creek watershed in northwest Indiana drains into Lake Michigan near several heavily used recreational beaches. This study aimed to investigate the levels of fecal indicator bacteria, enterococci and Bacteroidales, in Salt Creek using real-time quantitative PCR (qPCR) analysis and to obtain a preliminary assessment of the contribution of these organisms in effluent discharged by a publicly owned treatment works (POTW) into this stream. Bacterial concentrations were estimated from qPCR results as calibrator cell equivalents (CCE) at 1 site upstream and 5 sites downstream from the POTW as well as in the POTW effluent. Additionally, enterococci were enumerated on mEI media to compare CCE with culturable results. Our data indicated that the qPCR CCE/100 ml geometric mean over an 8 week collection period in the summer of 2007 ranged from 1.0 x 103 to 3.5 x 103 enterococci and from 3.6 x 104 to 2.3 x 105 total Bacteroidales at the 6 different stream sites. Analysis of Bacteroides thetaiotaomicron resulted in levels ranging from 8.9 x 102 to 9.0 x 103 at the same sites. The geometric mean concentrations of enteroccoci and Bacteroidales CCE in the effluent samples were approximately 1 log greater than the respective mean concentrations in the creek. Culture counts revealed that the geometric mean enterococci at these stream sites ranged from 5.0 x 102 to 2.6 x103 CFU/100 ml. Comparison of the trend between enterococci culture and qPCR data showed that the only major divergence was in the treated effluent where the mean CCE estimate was 3.1 x 104 CCE/100 ml and the mean culture concentration was 3.4 x 101 CFU/100 ml. Indicator levels in the stream samples collected right above and below the POTW were not significantly different for qPCR determined enterococci but were significantly greater below the outfall for Bacteroidales and above the outfall for culturable enterococci. Further studies are needed to evaluate the relationships between qPCR and culturable indicator levels in POTW effluents and receiving waters.

PRESENTATION Evaluation of a Real-Time Quantitative Pcr Method With Propidium Monazide Treatment for Analyses of Viable Fecal Indicator Bacteria in Wastewater Samples 06/01/2008
VARMA, M., R. FIELDS, M. K. STINSON, B. Rukovets, E. C. CHERN, L. J. WYMER, AND R. A. HAUGLAND. Evaluation of a Real-Time Quantitative Pcr Method With Propidium Monazide Treatment for Analyses of Viable Fecal Indicator Bacteria in Wastewater Samples. Presented at 2008 American Society for Microbiology General Meeting, Boston, MA, June 01, 2008 - June 05, 2208.
Abstract: The U.S. EPA is currently evaluating rapid, real-time quantitative PCR (qPCR) methods for determining recreational water quality based on measurements of fecal indicator bacteria DNA sequences. In order to potentially use qPCR for other Clean Water Act needs, such as updating criteria for disinfected POTW effluents, the ability to distinguish live organisms would be desirable. Propidium monoazide (PMA) has been shown to be selective in penetrating the cellular membranes of dead microorganisms where, upon exposure to light, it renders the DNA sequences in these cells unavailable for PCR detection. Therefore only sequences from live organisms should be measured by qPCR following PMA treatment. In this study a qPCR method incorporating pretreatment of samples with PMA was evaluated using pure cultures and wastewater samples collected from 3 different POTWs at 4 treatment stages including the chlorine-disinfected effluents. Treatment of heat-killed, cultured Enterococcus faecalis and Bacteroides thetaiotaomicron cells with PMA resulted in ~3-4 log reductions in QPCR-detectable target sequences compared with levels detected from live cells exposed to PMA or killed cells that were not exposed to PMA. Similar results were seen with cell concentrates from 1 ml buffer and wastewater samples spiked with E. faecalis, however, an inhibitory effect on PMA activity was indicated when 10ml of the wastewater samples were processed. Analyses of wastewater samples collected from the POTWs during normal dry weather operation showed that culturable Enterococcus and fecal coliform bacteria counts were more closely associated with QPCR-determined levels of target sequences from Enterococcus and Bacteroidales in PMA treated samples than with those in samples without PMA treatment. This trend was not as evident in samples collected from the same plants during wet weather operation where blending of primary and secondary-treated samples occurred prior to disinfection. Further studies are needed to determine the efficacy of PMA treatment for distinguishing viable organisms in different wastewater and surface water matrices by qPCR analysis.

PRESENTATION In-Vitro Cell Culture and Real-Time Reverse Transcriptase Pcr-Based Assays to Detect Infective Toxoplas Gondii Oocysts 06/01/2008
AUGUSTINE, S. J., L. F. VILLEGAS, M. W. WARE, S. L. HAYES, M. J. See, J. P. Dubey, AND E. VILLEGAS. In-Vitro Cell Culture and Real-Time Reverse Transcriptase Pcr-Based Assays to Detect Infective Toxoplas Gondii Oocysts. Presented at 2008 American Society for Microbiology Annual Meeting, Boston, MA, June 01, 2008.
Abstract: Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canada and Brazil have shown this parasite to also cause waterborne outbreaks of toxoplasmosis, suggesting its presence and persistence in drinking and recreational waters. To date, little is known about the prevalence of T. gondii oocysts in water and its resistance to disinfection. In this study, we developed a quantitative reverse-transcriptase PCR (RT-qPCR) assay using four constitutive and inducible gene targets (gra7, act1, sporosag, and tgowp genes) to detect and quantify viable T. gondii oocysts. Additionally, an in vitro cell culture assay was also developed to determine viability of T. gondii. Preliminary results revealed that although sporosag is specifically expressed in the infective sporulated oocyst stage, levels of sporosag mRNA did not correlate with viability. Similar results were obtained with gra7, act1, and Tgowp gene targets. In contrast, our in vitro cell culture assay provided a sensitive and quantitative assay to measure disinfection treatment efficacies commonly used to inactivate waterborne parasites. Initial results indicated that oocysts treated with 10% formalin or heat inactivation for 1 hour at 80oC were effectively killed. Other treatments that had marked effects include sodium hypochlorite, Wescodyne, ethanol, and UV. This study reveals that mRNA-based PCR viability assay using gra7, act1, sporosag, and tgowp genes may not be useful gene candidates to measure potential infectivity of T. gondii oocysts. Other mRNA species that have a shorter half-life may prove more useful. We also present a novel in vitro cell culture assay that can be used to quantify treatment efficacies commonly used in drinking and wastewater industries.

PRESENTATION Laboratory Evaluations of the Enterococcus Qpcr Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements 06/01/2008
HAUGLAND, R. A., S. SIEFRING, M. VARMA, AND L. J. WYMER. Laboratory Evaluations of the Enterococcus Qpcr Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements. Presented at 108th General Meeting of the American Society for Microbiology, Boston, MA, June 01 - 05, 2008.
Abstract: The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for enterococci that meets the requirements for rapid, risk-based water quality assessments at beaches. However, questions remain over how this method should be used in water quality criteria. These questions relate in part to the lack of a clear understanding of the performance characteristics of the method such as detection limits and precision under the influence of mixed sources of variation. In the present study, single laboratory results are reported that further define these attributes. The 95% confidence method detection limit (95% MDL) was estimated at ~100 calibrator cell equivalents (CE)/sample from analysis results of replicate 100 ml buffer samples spiked with 10-1000 cultured E. faecalis cells. This CE value can be translated to a 95% MDL of 3230 target sequence copies (TSC)/sample based on the recovery efficiency of 32 TSC/cell determined for these samples or 5.4 TSC/reaction based on the use of 0.166% of the sample extracts for PCR analysis. Pure method precision, expressed as log10 variance, was estimated as 0.018, 0.054 and 0.114 from spikes of 1000, 333 and 100 cells, respectively. Analyses of diverse surface water samples indicated that the influence of non-interfering matrices on log10 variance was negligible. Based on analyses of different sets of beach water samples from 2003-2005 U.S. EPA epidemiological studies where the geometric means for ambient enterococci by qPCR were within 100 to 1000 CE, the 90% prediction interval was estimated as 170 to 650 for a true value of 333 CE. These analyses further indicated that the major source of the measurement uncertainty was associated with sample variability. Accurate characterization of beach water quality by this method is therefore highly dependent upon the analysis of multiple samples.

PRESENTATION A New Electropositive Filter for Concentrating Enterovirus and Norovirus from Large Volumes of Water Mceard 06/01/2008
Karim, M., E. RHODES, N. BRINKMAN, L. J. WYMER, AND G. FOUT. A New Electropositive Filter for Concentrating Enterovirus and Norovirus from Large Volumes of Water Mceard. Presented at ASM Annual Meeting, Boston, MA, June 01 - 05, 2008.
Abstract: The detection of enteric viruses in environmental water usually requires the concentration of viruses from large volumes of water. The 1MDS electropositive filter is commonly used for concentrating enteric viruses from water but unfortunately these filters are not cost-effective for routine viral monitoring. In this study, an inexpensive electropositive cartridge filter, the Nanoceram® filter, was evaluated for its ability to concentrate enterovirus and norovirus from large volumes of water. In initial experiments, one hundred liters of deionized water was seeded with 105 PFU of poliovirus 1 and concentrated using the same adsorption-elution procedure used for the 1 MDS filter. The mean retention of seeded poliovirus by Nanoceram® filters was 84 percent. To optimize the elution procedure, six protocols, each comprised of two successive elutions with varying lengths of filter emersion, were evaluated. There was a significant difference among the elution procedures tested (p=0.025). The highest virus recovery (77%) was obtained by immersing the filters in beef extract for 1 min during the first elution and 15 minutes during the second elution. The recovery efficiencies of poliovirus, coxsackie B5, and echovirus 7 from 100 liters of seeded tap water were 54%, 27%, and 32%, respectively. Finally, virus recoveries by Nanoceram® filters were compared with 1MDS filters using tap water and Ohio River water. The mean recoveries of poliovirus from 100L of tap water using Nanoceram® and 1MDS filters were 51% and 67%, respectively, and the recoveries from Ohio River water were 38% and 36%, respectively. The recoveries of Norwalk virus by Nanoceram® filters from tap and river water was higher than the recoveries by 1MDS filters. These data suggest that Nanoceram® filters can be used as an inexpensive alternative of 1MDS filters for routine viral monitoring of water.

PRESENTATION Rna Extraction Methods for Real-Time Pcr and Microarray Analyses of Cryptosporidium and Toxoplasma Gondii Oocysts 2nd Presentation 05/28/2008
See, M. J., M. W. WARE, J. P. Dubey, AND E. VILLEGAS. Rna Extraction Methods for Real-Time Pcr and Microarray Analyses of Cryptosporidium and Toxoplasma Gondii Oocysts 2nd Presentation. Presented at 10th International Workshop on Opportunistic Protists, Boxton, MA, May 28 - 31, 2008.
Abstract: The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion processes or about their survival after disinfection treatment. Microarrays and reverse transcriptase real-time PCR (RT-qPCR) are useful techniques that are amenable to identifying genes involved in regulating such mechanisms. In order to effectively perform these analyses, high quality RNA must be isolated. Here, we evaluated four RNA extraction methods (Ambion, RiboPure, Qiagen RNeasy, Epicentre Master Pure and TRIzol LS reagent) in their ability to extract high quality RNA from C. parvum and T. gondii oocysts. RNA quality was measured and expressed as an RNA Integrity Number (RIN), with a 9.0 or above indicating minimal degradation, while RNA purity was determined using RT-qPCR. Preliminary results indicated high quality RNA (RIN 8.3 – 9.8) was extracted from C. parvum oocysts using all four extraction methods; however, genomic DNA (gDNA) was present in all samples. DNase I treatment effectively removed gDNA contaminants only in Ambion and Qiagen extracted RNA, but the quality was compromised (RIN 5.5 - 7.7). RNA extracted from T. gondii oocysts proved to be more difficult and the quality/purity of RNA extracted was variable. Nevertheless, all RNA extraction methods used in this study were effective in quantitating hsp-70 and β-tubulin expression in C. parvum oocysts and SporoSAG expression in T. gondii oocysts using RT-qPCR. These studies will now enable us to use RT-qPCR, and perhaps microarray analysis, for identifying novel genes essential for oocyst development, survival in the environment, and the establishment of infection in the host.

PRESENTATION Cryptosporidium Source Tracking in the Potomac River Watershed 05/28/2008
Wang, W., P. Chen, E. VILLEGAS, R. B. LANDY, C. KANETSKY, V. Cama, T. Dearen, C. Schultz, K. G. Orndorff, G. J. Prelewwicz, M. H. Brown, AND K. R. Young. Cryptosporidium Source Tracking in the Potomac River Watershed. Presented at 10th International Workshop on Opportunistic Protists, Boston, MA, May 28 - 31, 2008.
Abstract: To better characterize the presence of Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base-flow and 28 storm-flow samples from five sites within the watershed. These sites included two water treatment plant intakes as well as three upstream sites, each associated with a different type of land use. Each of these uses, urban/wastewater, agricultural (cattle)/wastewater, and agricultural (cattle), posed different risks with regard to the potential contribution of Cryptosporidium oocysts to the source water. In the study, Cryptosporidium was detected in 27 base-flow water samples and 23 storm-flow water samples. The most frequent genotype detected was C. andersoni (detected in 41 samples), while 14 other species/genotypes, almost all wildlife-associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites under agricultural influence, it was largely absent at the urban/wastewater site. There were very few positive samples by EPA Method 1623 at any site; only eight of 90 samples (9%) analyzed were positive for Cryptosporidium by microscopy. The genotyping results suggest that much of the Cryptosporidium that is present in the water treatment plant source waters may not pose a significant human health risk.

PRESENTATION Analysis of Arsenical and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS 05/21/2008
KUBACHKA, K., C. GALLAWA, P. A. CREED, J. T. CREED, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Analysis of Arsenical and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS. Presented at 2nd International Congress: Arsenic in the Environment, Valencia, SPAIN, May 21 - 23, 2008.
Abstract: Slide presentation at the 2nd International Congress: Arsenic in the Environment, Valencia, Spain, May 21-23, 2008

PRESENTATION The Role of Biofilms in Drinking Water Exposure to Potentially Pathogenic Legionella Spp. 05/20/2008
LAU, H. Y. The Role of Biofilms in Drinking Water Exposure to Potentially Pathogenic Legionella Spp. Presented at 2008 EPA Science Forum, Washington, DC, May 20 - 22, 2008.
Abstract: Legionellosis is a bacterial infection caused by species of the genera Legionella and is the most common waterborne disease reported in the United States. This type of infection has two clinically distinct forms: Legionnaire's disease, a severe type of infection, which includes pneumonia, and Pontiac fever, a milder self-limiting illness. Legionella spp. are unbiquitous in nature with water being the major reservoir for these organisms. Exposure to Legionella spp. occurs via the inhalation of contaminated aerosols from devices such as cooling towers, showers and faucets. In these artificial water systems, Legionella spp. growth is detected almost exclusively in nutrient-rich biofilms covering the inerior of pipe walls, in-premises plumbing fixtures and heating, ventilation, and air-conditioning systems. The types of disinfectant residuals in drinking water systems have been shown to directly influence both the composition of biofim communities and on biofilm development. Thus drinking water systems not only serve as a reservoir for potential pathogens entering the system, their interactions and development ini biofilm communities has the potential to select for stains that are more fit to thrive in this type of environment. Therefore, understanding how Legionella spp. propagates and persists in man-made water systems, and the role biofilms play in that process, is critical to the development of strategies that can more effectively control their occurrence in drinking water.

PRESENTATION The Enterococci Qpcr Method for Recreational Water Quality Testing: Background and Single Lab Performance 05/13/2008
HAUGLAND, R. A. The Enterococci Qpcr Method for Recreational Water Quality Testing: Background and Single Lab Performance. Presented at Scientific Meeting on Development, Validation and Implementation of qPCR and PCR Methods for Use in Recreational Waters, Cincinnati, OH, May 13, 2008.
Abstract: Slide Presentation given at the Scientific Meeting on Development, Validation and Implementation of qPCR and PCR Methods for Use in Recreational Waters

PRESENTATION Microbial Risk Assessment: Underpinning Research Needs for Recycled Water 05/05/2008
ASHBOLT, N. Microbial Risk Assessment: Underpinning Research Needs for Recycled Water. Presented at 12th Annual Water Reuse and Desalination Research Conference, Denver, CO, May 05 - 06, 2008.
Abstract: Slide Presentation to be presented at the 12th Annual Water Reuse and Desalination Research Conference, Denver, Colorado, May 5-6, 2008

PRESENTATION Improving Lab Sample Management Pos/Mceard 04/23/2008
BASSETT, M. AND M. BINFORD. Improving Lab Sample Management Pos/Mceard. Presented at 2008 EPA 2008 Conference on Managing Environmental Quality Systems, Seattle, WA, April 21 - 24, 2008.
Abstract: "Scientists face increasing challenges in managing their laboratory samples, including long-term storage of legacy samples, tracking multiple aliquots of samples for many experiments, and linking metadata to these samples. Other factors complicating sample management include the need to share samples amongst team members and dealing with multiple sample storage units. To address these issues, the National Exposure Research Laboratory in Cincinnati has an ongoing project to increase scientist’s productivity and improve the condition under which their samples are stored. This talk will provide details of the project, including the development of a sample handling policy and the deployment of an electronic sample tracking system."

PRESENTATION Quantitative Microbial Risk Assessment of Drinking Water the Next Step 04/16/2008
ASHBOLT, N. Quantitative Microbial Risk Assessment of Drinking Water the Next Step. Presented at Meeting at the Technische Universiteit Delft, Holland, NETHERLANDS, April 16, 2008.
Abstract: Presentation given at the Technische Universiteit Delft, Holland, The Netherlands, April 16, 2008

PRESENTATION Feasibility of Community Food Item Collection for the National Children's Study 04/16/2008
Jordan, K. C., M. Knuth, B. Sherwood, R. R. Larson, L. J. MELNYK, S. McNutt, J. J. QUACKENBOSS, S. M. Viet, AND L. J. Moyer-Mileur. Feasibility of Community Food Item Collection for the National Children's Study. Presented at Utah Dietetic Association - University of Utah Research Conference, Salt Lake City, UT, April 16 - 18, 2008.
Abstract: The National Children’s Study proposes to investigate the role of contaminants on health outcomes in pregnant women and children. A specific area of concern is contaminant exposure through the ingestion of solid foods. National food contaminant databases may miss environmental exposures unique to specific communities. Therefore, alternative sampling procedures are proposed to capture contributing environmental exposures on a household level, such as storage, preparation, and consumption in the residence.

PRESENTATION Development of a Microarray Detection Method for Waterborne Pathogens Mceard/Barb 04/08/2008
BRINKMAN, N., E. VILLEGAS, R. Francisco, F. W. SCHAEFER, T. NICHOLS, D. Roberts, AND P. Schaudies. Development of a Microarray Detection Method for Waterborne Pathogens Mceard/Barb. Presented at Joint EPA and DHS Conference on Real-World Applications and Solutions for Microbial Risk Assessment, Bethesda, MD, April 08 - 10, 2008.
Abstract: Presentation at the Joint EPA and DHS Conference on Real-World Applications and Solutions for Microbial Risk Assessment in Bethesda, MD, April 8-10, 2008

PRESENTATION Mra Research Activities to Support Drinking Water Standards 04/08/2008
ASHBOLT, N. Mra Research Activities to Support Drinking Water Standards. Presented at The Joint US.EPA and Department of Homeland Security Conference on Real-World Applications and Solutions for Microbial Risk Assessment, Bethesda, MD, April 08 - 10, 2008.
Abstract: Slide Presentation for the Joint U.S. EPA and Department of Homeland Security Conference on Real-World Applications and Solutions for Microbial Risks Assessment, Bethesda, MD, April 8-10, 2008

PRESENTATION Rna Extraction Methods for Reverse Transcriptase Real-Time Pcr and Microarray Analysis of Cryptosporidium and Toxoplasma Gondii Oocysts 03/29/2008
See, M. J., M. W. WARE, J. P. Dubey, AND E. VILLEGAS. Rna Extraction Methods for Reverse Transcriptase Real-Time Pcr and Microarray Analysis of Cryptosporidium and Toxoplasma Gondii Oocysts. Presented at ASM Annual Meeting, Muncie, IN, March 28 - 29, 2008.
Abstract: The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion processes or about their survival after disinfection treatment. Microarrays and reverse transcriptase real-time PCR (RT-qPCR) are useful techniques that are amenable to identifying genes involved in regulating such mechanisms. In order to effectively perform these analyses, high quality RNA must be isolated. Here, we evaluated four RNA extraction methods (Ambion RiboPure, Qiagen RNeasy, Epicentre Master Pure and TRIzol LS reagent) in their ability to extract high quality RNA from C. parvum and T. gondii oocysts. RNA quality was measured and expressed as an RNA Integrity Number (RIN), with a 9.0 or above indicating minimal degradation, while RNA purity was determined using RT-qPCR. Preliminary results indicated high quality RNA (RIN 8.3 – 9.8) was extracted from C. parvum oocysts using all four extraction methods; however, genomic DNA (gDNA) was present in all samples. DNase I treatment effectively removed gDNA contaminants only in Ambion and Qiagen extracted RNA, but the quality was compromised (RIN 5.5 - 7.7). RNA extracted from T. gondii oocysts proved to be more difficult and the quality/purity of RNA extracted was variable. Nevertheless, all RNA extraction methods used in this study were effective in quantitating hsp-70 and β-tubulin expression in C. parvum oocysts and SporoSAG expression in T. gondii oocysts using RT-qPCR. These studies will now enable us to use RT-qPCR, and perhaps microarray analysis, for identifying novel genes essential for oocyst development, survival in the environment, and establishment of infection in the host.

PRESENTATION Molecular Genotyping of Viable Cryptosporidium Oocysts 03/28/2008
Brescia, C., M. W. WARE, S. L. BARTELT-HUNT, A. EGOROV, AND E. VILLEGAS. Molecular Genotyping of Viable Cryptosporidium Oocysts. Presented at ASM Annual Meeting, Muncie, IN, March 28 - 29, 2008.
Abstract: Cryptosporidium is a chlorination-resistant protozoan parasite that causes a self-limiting diarrheal disease in the immunocompetent or severe chronic diarrhea in the immunocompromised. Two species, C. parvum and C. hominis, cause most cases of cryptosporidiosis in humans, while C. muris infects primarily rodents. The current US EPA standard methods for the detection of Cryptosporidium oocysts in water, Methods 1622 and 1623, are labor intensive and cannot determine the species or distinguish viable from nonviable oocysts. The goal of this project is to develop a rapid molecular method that specifically detects viable waterborne Cryptosporidium oocysts and that determines the species present in a sample. This alternative technique employs propidium monoazide (PMA) staining in conjunction with conventional PCR detection and genotyping. PMA is a DNA intercalating dye that only penetrates dead cells. Using traditional DNA extraction procedures, only the genomic DNA from live cells, which is not intercalated with PMA, is retained and detected in subsequent PCR analysis. To date, PMA has only been used to differentiate live/dead bacteria and fungi. It has not yet been applied to protozoan parasites. Preliminary results revealed that heat killed oocysts treated with PMA were not detected while live oocysts treated with PMA were detected using conventional PCR. This method was then paired with Cryptosporidium PCR-based genotyping methods to allow for species discrimination. When live C. parvum and dead C. muris oocysts were mixed at a 1:9 ratio, only live C. parvum oocysts were detected. These results indicate that PMA treatment is very specific at detecting DNA from live oocysts in the presence of a higher concentration of dead oocysts. We are currently investigating the application of this method in environmental water matrices.

PRESENTATION Methods for Measuring Occurrence and Exposure from Viruses in Drinking and Recreational Water 03/25/2008
FOUT, G., N. BRINKMAN, M. Karim, E. RHODES, A. EGOROV, AND S. L. BARTELT-HUNT. Methods for Measuring Occurrence and Exposure from Viruses in Drinking and Recreational Water. Presented at Future Develiopment Director for Safe Drinking Water Management International Seminar, Incheon, SOUTH KOREA, March 25, 2008.
Abstract: The United States Environmental Protection Agency (EPA) has an active research program to develop and improve methods for detecting human enteric viruses in recreational, source, and drinking waters. EPA is also developing methods to measure exposure to waterborne viruses and applying these methods to studies to assess health benefits associated with EPA drinking water rules. This paper describes EPA’s recent research on three methodologies in these areas. First, in an effort to reduce the costs associated with virus occurrence studies, EPA evaluated the NanoCeram™ filter, a new positively charged cartridge filter for concentrating viruses from water. EPA’s results showed that this filter is equivalent to the Virosorb® 1MDS cartridge filter that is specified for use in the standardized total culturable virus assay from EPA’s Information Collection Rule. Second, EPA conducted a proof of concept study to demonstrate the usefulness of a generic microarray format for virus identification. This study showed that the generic microarray can accurately type noroviruses. Third, EPA has developed a microbead immunoassay for detection of salivary antibodies against potential waterborne pathogens, including noroviruses, rotaviruses, Cryptosporidium, Toxoplasma gondii, and Helicobacter pylori.

PRESENTATION Emerging Contaminants in the Drinking Water Cycle Mceard 01/29/2008
GLASSMEYER, S. Emerging Contaminants in the Drinking Water Cycle Mceard. Presented at Presentation at Wooster College, Wooster, OH, January 29, 2008.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-g/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the complex mixtures of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. In the source water of one conventional drinking water facility, 45 out of 113 monitored chemicals were detected at least once, with 21 chemicals still detectable in the finished drinking water. This documents the incomplete removal of such chemicals during treatment. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Investigation of the Biotransformation of a Dma in Mouse Cecum Samples Using Ic-ICP-MS and LC-Esi-MS/MS Detection 01/06/2008
KUBACHKA, K., S. CONKLIN, J. T. CREED, AND D. J. THOMAS. Investigation of the Biotransformation of a Dma in Mouse Cecum Samples Using Ic-ICP-MS and LC-Esi-MS/MS Detection. Presented at 2008 Winter Conference on Plasma Spectrochemistry, Tennecula, CO, January 06 - 12, 2008.
Abstract: Recent arsenic metabolism studies have begun to indicate the presence of sulfur analogs of the more common arsenic oxides in biological systems. An emerging area of research is how and where these arsenic species are formed in the metabolic pathway. The authors have previously indicated that the microflora in the cecum of a mouse is able to biotransform an arsenosugar oxide (a tri-alkyl substituted arsenic oxide) to its corresponding sulfide [1]. The initial mechanism of the aforementioned conversion theorized that the bacteria produced H2S as a waste product which then facilitated the conversion. Subsequent research using H2S in an ideal solution indicated that the rate of conversion was influenced by the degree of substitution of the arsenic oxide. For instance, the reaction rate for tetramethylarsine oxide (TMAO, tri-alkyl substituted) was faster than dimethylarsinic acid (DMA, di-alkyl substituted) which was faster than monomethylarsinic acid (MMA, mono-alkyl substituted) [2]. The conversion of DMA to dimethylthioarsinic acid (DMTA) in these ideal solutions raised the question if a similar conversion would be observed in the anaerobically incubated cecal samples from a mouse. In this experiment, DMA was added at various concentrations to the cecum contents of a mouse, which were then incubated at 37 °C. The DMA spiked cecum samples were incubated for various periods and then flash frozen at -80 °C to minimize additional unwanted activity and subsequent transformation. An outline of the study design is given below: Cecum Samples (cecum + buffer) Time (Hours at 37 °C) Arsenic (ng/g) 0 1 6 18 24 0 X X X 20 X X X X X 200 X X X X X 1000 X X Controls (cecum, no buffer) Time (Hours) Arsenic (ng/g) 0 24 0 X X 20 X X 200 X X 1000 X X The fortified samples, blanks and controls were analyzed for arsenic metabolites via speciation with IC-ICP-MS, utilizing arsenic specific detection at m/z 75. Molecular characterization was also conducted using LC-ESI-MS/MS. The metabolites detected were: DMA, DMTA, dimethyldithioarsinic acid (DMDTA), and trimethylarsine sulfide (TMAS). Both elemental and molecular data will be presented to support these identifications. The presence of TMAS was not expected and thorough analysis of controls and blanks lead the authors to believe it is not an artifact of sample contamination. Its presence could possibly be explained by DMA being methylated to TMAO, which could then be sulfurylated to TMAS. Finally, the conversion from DMA to the corresponding sulfur analogs will be presented with respect to incubation times. This data set indicates that sulfur analogs of arsenic oxides may be produced in the gastrointestinal tract. The data set shows evidence of novel biotransformation pathways of DMA in the gastrointestinal tract, and should prove useful in further understanding the arsenic metabolism pathways.

PRESENTATION Presence of Pharmaceuticals in Groundwater Down Gradient from Wastewater Lagoons Receiving Partially Treated Wastewater 11/11/2007
GLASSMEYER, S., C. Kenah, E. T. Furlong, AND D. Kolpin. Presence of Pharmaceuticals in Groundwater Down Gradient from Wastewater Lagoons Receiving Partially Treated Wastewater. Presented at SETAC North American 28th Annual Meeting, Milwaukee, WI, November 11 - 15, 2007.
Abstract: Wastewater can contain traces of the pharmaceutical compounds that are used within a given household or community. These chemicals can act as markers of the wastewater, and their presence can help determine potential sources of contamination of water resources. For this study, groundwater samples were collected up and down gradient from an unlined wastewater treatment plant lagoon serving 400 households. All samples were analyzed for 16 pharmaceuticals using liquid chromatography/ mass spectrometry. The study consisted of two phases. In Phase I (August 2005), 13 different boring locations were drilled, and samples were collected at two to three depths within each hole to determine the boundaries of the leachate plume. In addition, the treated effluent, the municipal lagoon, and the community’s drinking water were also sampled. Monitoring wells (MW) were installed adjacent to three of the bore holes, one up gradient, and two down gradient. During Phase II, the three MWs, the lagoon, and the drinking water source were sampled quarterly (January, April, July and October 2006) to examine seasonal trends in chemical concentrations. Thirteen of the 16 target pharmaceutical compounds were detected at least once. Carbamazepine and sulfamethoxazole were the most commonly detected, occurring in every sample collected from the two down gradient MWs, The carbamazepine concentrations in the MW proximate to the lagoon (MW 2) had concentrations which ranged from 0.316 to 0.642 µg/L, median 0.513 µg/L, which were greater than the concentrations we measured in the lagoon samples (0.02 to 0.557 µg/L, median 0.044 µg/L). The concentrations in MW 2 were even greater than the treated wastewater effluent samples collected in 2002 (max = 0.270 µg/L). The samples collected from multiple depths within each boring location exhibited the highest concentrations of pharmaceuticals in the shallower samples (25-45 feet below the surface), with decreasing cocentrations with increasing depths.

PRESENTATION Evaluation of a Microarray for Genotyping Noroviruses 11/10/2007
FOUT, G. AND N. BRINKMAN. Evaluation of a Microarray for Genotyping Noroviruses. Presented at Third International Calcivirus Conference, Cancun, MEXICO, November 10 - 13, 2007.
Abstract: Noroviruses that infect humans are divided into three genogroups based upon their sequence diversity. Of these, genogroups I and II have been identified as leading causes of waterborne disease outbreaks worldwide and are frequently found in rivers and lakes that serve as drinking water sources. To develop a better understanding of the risk posed by these organisms, a more rapid procedure for identifying and genotyping noroviruses in the environment was investigated. Currently, noroviruses are usually detected in water samples using reverse transcriptase-polymerase chain reaction (RT-PCR) based assays. Although sequencing of RT-PCR amplicons is the gold standard for genotyping these viruses, RT-PCR assays of water concentrates often produce a range of non-specific amplicons in a single reaction. This usually necessitates the cloning of products prior to sequencing. To determine whether genotyping could be performed more rapidly and without the need for cloning, we evaluated the use of a generic microarray format using multiple strain specific probes that would be able to hybridize to region B of the RNA polymerase gene. With this method, a single base extension (SBE) reaction was used to label genotype-specific probes that had annealed to a matching virus sequence. This labeling was done in the presence of single and multiple norovirus templates. These probes were then hybridized to an Affymetrix GenFlex Tag Array and the labeled probes were detected. The specificity of this approach was examined by designing probes with and without nucleotide mismatches. Our results show that the perfect-matched tag-probes were specifically labeled in SBE reactions for all strains examined. In addition, most of the mismatched tag-probes were not labeled and those that were resulted in specific fingerprints that could be used to subtype strains. We also showed that norovirus seeded into source water concentrates can be genotyped. These results demonstrate the utility of the GenFlex Tag Array for genotyping noroviruses from water samples. This approach also could be adapted to include the simultaneous identification of multiple waterborne pathogens.

PRESENTATION Frank and Opportunistic Pathogen Exposure Risks from Distribution System Biofilms 11/09/2007
ASHBOLT, N. Frank and Opportunistic Pathogen Exposure Risks from Distribution System Biofilms. Presented at Meeting at Columbia University, New York, NY, November 09, 2007.
Abstract: To be presented at Columbia University, New York, NY, November 9, 2007

PRESENTATION Identification of Mycobacterium Avium Subsp. Hominissuis Isolated from Drinking Water 11/04/2007
KING, D. N., A. Beumer, AND S. L. PFALLER. Identification of Mycobacterium Avium Subsp. Hominissuis Isolated from Drinking Water. Presented at Water Technology Conference, Charlotte, NC, November 04 - 08, 2007.
Abstract: Mycobacterium avium (MA) is divided into four subspecies based primarily on host-range and consists of MA subsp. avium (birds), MA subsp. silvaticum (wood pigeons), MA subsp. paratuberculosis (broad, poorly-defined host range), and the recently described MA subsp. hominissuis (humans and swine). MA are ubiquitous in the environment and evidence suggests water is a possible source of human exposure. Routes of exposure to waterborne pathogens include ingestion, inhalation of water vapor, and ingestion of produce irrigated or washed with contaminated water. The goal of this study was to determine how specific MA subspecies hominissuis is to humans by subspeciating human clinical isolates of MA and also to determine the proportion of MA isolated from food and water that belong to this subspecies. Understanding the occurrence of MA subsp. hominissuis in water and food will aid the U.S. Environmental Protection Agency in assessing the risk of human exposure to these sources. Temperature growth range, IS1245 copy number, sequencing of the 16S-23S internal transcribed spacer region or hsp65 gene and other methods have been used to differentiate subspecies hominissuis from the other subspecies. MA subsp. hominissuis can grow on Lowenstein Jensen (LJ) medium supplemented with pyruvate at 45 ۫C while bird type isolates cannot (Mijs et al., 2002). Clinical and environmental MA isolates from our collection were inoculated on this medium and allowed to incubate for up to 90 days. Ninety percent of the isolates grew at 25 ۫C and 67% grew at 45 ۫C. Isolates that grew at 45 ۫C also grew at 25 ۫C. Nearly 100% of the human isolates included in this study grew at 45 ۫C, suggesting they are strains of MA subsp. hominissuis. In addition, we performed PCR and sequence analysis on the 3′ end of the hsp65 gene as a means to confirm MA subsp. hominissuis identification. Human isolates had hsp65 sequences closely related to hsp65 sequences from MA subspecies hominissuis described previously and several hsp65 sequences from environmental isolates were identical or very closely related to those of hominissuis. None of the human or environmental isolates in our study had hsp65 sequences that clustered with MA subsp. avium or paratuberculosis. These data suggest treated drinking water and produce are potential sources of exposure to the type of MA that can infect humans.

PRESENTATION Investigation of Reagent Gases for the Positive Chemical Ionization of the Polybrominated Diphenyl Ethers 10/18/2007
PAWLECKI-VONDERHEIDE, A. M., T. E. HIEBER, P. KAUFFMAN, J. N. MORGAN, AND L. J. MELNYK. Investigation of Reagent Gases for the Positive Chemical Ionization of the Polybrominated Diphenyl Ethers. Presented at 2007 Federation of Analytical Chemistry & Spectroscopy Societies (FACSS) Conference, Memphis, TN, October 14 - 18, 2007.
Abstract: To be presented at the 2007 Fedration of Analytical Chemistry and Spectroscopy Societies (FACSS) Conference, October 14-18, 2007, Memphis, TN

PRESENTATION The Use of Technologies: Exposure (Cross Contamination), Risk Assessment, and Guidelines 10/17/2007
ASHBOLT, N. The Use of Technologies: Exposure (Cross Contamination), Risk Assessment, and Guidelines. Presented at Institute of Medicine Rountable: Global Environmental Health, Washington, DC, October 17 - 18, 2007.
Abstract: Presentation given at the Institute of Medicine Roundtable: Global Environmental Health, Washington, DC, October 17-18, 2007

PRESENTATION Incidental Water Ingestion During Recreational Swimming 10/15/2007
DUFOUR, A. P. Incidental Water Ingestion During Recreational Swimming. Presented at 2007 International Society for Exposure Analysis Annual Meeting, Raleigh, NC, October 15 - 18, 2007.
Abstract: To be presented at the 2007 International Society for Exposure Analysis Annual Meeting

PRESENTATION Monitoring of Microbes in Drinking Water 10/15/2007
ASHBOLT, N. Monitoring of Microbes in Drinking Water. Presented at 2007 International Society for Exposure Analysis Annual Meeting, Raleigh, NC, October 15 - 18, 2007.
Abstract: Internationally there is a move towards managing the provision of safe drinking water by direct assessment of the performance of key pathogen barriers (critical control points), rather than end point testing (i.e. in drinking water). For fecal pathogens that breakthrough the various barriers in a drinking water supply system, a residual chlorine concentration may provide further health protection. However, short-term failures (the most likely in systems) are likely to overwhelm the chlorine residual. Of even greater concern is the view that short-term failures are unlikely to be detected by either compliance-type routine monitoring of drinking waters or by pathogen-specific monitoring, such as monthly and more frequent for Cryptosporidium oocysts. On the other hand, a rapid drop in chlorine residual (preferably measured on-line) provides a good trigger for follow-up investigation, well ahead of results from routine microbiological assessment. An emerging issue, however, is the role of distribution system biofilms, which are ubiquitous and a potential site for pathogen accumulation and growth post water treatment. Biofilms are largely extracellular bacterial mucilage that can protect the microbial inhabitants from a residual disinfectant. Further, amoeba that predate on biofilm bacteria are known hosts for the selection of opportunistic bacterial pathogens, such as strains of Legionella, Mycobacterium and other pathogens. The role of biofilms/amoeba for antibiotic gene amplification/transfer has yet to be quantified. Hence, within the risk management paradigm for water system monitoring, assessing biofilm-pathogens deserves greater attention. In addition, we need to move on from only assessing diarrheal impact and include more severe and longer-lasting diseases (sequelae), such as arthritis and various cancers possibly caused by waterborne pathogens. In the future, perhaps we would be better protected by monitoring biofilms via a more metagenomic approach seeking to identify changes in community ecology and key genes? Views expressed are not necessarily those of the US-EPA.

PRESENTATION Surface-to-Food Pesticide Transfer as a Function of Fat and Moisture Content 10/14/2007
PAWLECKI-VONDERHEIDE, A. M., T. E. HIEBER, P. KAUFFMAN, J. N. MORGAN, AND L. J. MELNYK. Surface-to-Food Pesticide Transfer as a Function of Fat and Moisture Content. Presented at 2007 ISEA Meeting, Research Triangle Park, NC, October 14 - 18, 2007.
Abstract: To be presented at the 2007 ISEA Meeting, Durham/Research Triangle Park, NC October 14-18, 2007

PRESENTATION Community Duplicate Diet Methodology 10/14/2007
MELNYK, L. J., C. Clarke, S. McNutt, AND J. J. QUACKENBOSS. Community Duplicate Diet Methodology. Presented at ISEA Annual Meeting, Durham, NC, October 14 - 18, 2007.
Abstract: To be presented at the ISEA Annual Meeting in Durham, NC, October 14-18, 2007

PRESENTATION Statistical Framework for Recreational Water Quality Criteria and Monitoring, a New Title in the John Wiley & Sons Series, "STATISTICS in Practice" 10/03/2007
WYMER, L. J. Statistical Framework for Recreational Water Quality Criteria and Monitoring, a New Title in the John Wiley & Sons Series, "STATISTICS in Practice". Presented at 7th Annual Great Lakes Beach Association Meeting, Traverse City, MI, October 03 - 05, 2007.
Abstract: To be presented at the 7th Annnual Great Lakes Beach Association Meeting, October 3-5, 2007

PRESENTATION Statistical Framework for Recreational Water Quality Criteria and Monitoring 10/03/2007
WYMER, L. J. Statistical Framework for Recreational Water Quality Criteria and Monitoring. Presented at 7th Annual Great Lakes Beach Association Meeting, Cincinnati, OH, October 03 - 05, 2007.
Abstract: Discussion between the EPA Office of Research and Development (ORD) and the EPA Office of Water (OW), which is charged with setting criteria in accordance with the BEACH Act of 2000, have made it clear that in-depth statistical guidance for such criteria is needed. In January 2004, a workshop was conducted at ORD's research center in Cincinnati, Ohio, which attempted to address many of the statistical concerns expressed by OW and to develop an outline for a book on the subject. The demand for such a book was evident given that OW's questions were not unique, but were in fact being asked by all involved in assessing recreational water quality. Consequently, the book is aimed at public health officials, government regulators and the water microbiology science community in general. Contributors consist of preeminent leaders in environmental statistics and recreational water modeling. Individual chapters were written by those with expertise in the respective subject matter. European perspectives are included, along with those of the United States, Canada, and our colleagues "down under." Chapters are arranged in a logical progression covering the history of recreational water quality management, present-day management perspectives, rationale for the use of indicators, statistical process control and quality control concepts, sampling designs, uses of arithmetic and geometric means, case study in sampling, risk assessment modeling, concentration-response modeling, modeling and forecasting techniques as adjuncts to monitoring, and sensitivity analysis.

PRESENTATION The Enterococcus Qpcr Method for Recreational Water Quality Testing: Testing Background, Performance and Issues 10/03/2007
HAUGLAND, R. A. The Enterococcus Qpcr Method for Recreational Water Quality Testing: Testing Background, Performance and Issues. Presented at 7th Annual Great Lakes Beach Association Meeting, Traverse City, MI, October 03 - 05, 2007.
Abstract: Currently accepted culture-based monitoring methods for fecal indicator bacteria in surface waters take at least 24 hr to determine if unacceptable levels of fecal pollution have reached our recreational beaches. During this waiting period changing water conditions may result either in unnecessary beach closings or exposures to unsafe conditions. Newer molecular based technologies such as real-time quantitative PCR (QPCR) have the ability to provide measurements of fecal indicator bacteria nucleic acids within a few hours and thus could lead to more accurate and health protective advisories to the public due to their greater timeliness. The National Epidemiological and Environmental Assessment of Recreational (NEEAR) Waters Studies, performed by U.S. EPA and CDC in 2003-2005 demonstrated a correlation between swimming-related gastrointestinal illness rates and QPCR measurements of Enterococcus fecal bacteria at Great Lakes fresh water and Gulf Coast marine water beaches. These results suggest that the QPCR method can provide determinations of fecal pollution levels that are predictive of swimming-related health risks. This presentation will provide an overview of the Enterococcus QPCR method including a brief description of the method and its history, a synopsis of studies that have been conducted to determine its performance characteristics and a discussion of unresolved issues associated with its potential implementation on a national scale.

PRESENTATION Enteroccocci and Fecal Streptococci Membrane Filter Methods 09/19/2007
BRENNER, K. P. Enteroccocci and Fecal Streptococci Membrane Filter Methods. Presented at Microbiological Drinking Water Certification Training Course, Cincinnati, OH, September 19, 2007.
Abstract: To be presented at the Microbiology Drinking Water Certification Training Course, Cincinnati, OH, September 19, 2007

PRESENTATION Mi Medium 09/18/2007
BRENNER, K. P. Mi Medium. Presented at Microbiology Drinking Water Certification Training Course, Cincinnati, OH, September 18, 2007.
Abstract: To be presented at the Microbiology Drinking Water Certification Training Course, Cincinnati, OH, September 18, 2007

PRESENTATION Membrane Filter Methods 09/18/2007
BRENNER, K. P. Membrane Filter Methods. Presented at Microbiology Drinking Water Certification Training Course, Cincinnati, OH, September 18, 2007.
Abstract: To be presented at the Microbiology Drinking Water Certification Training

PRESENTATION Cryptosporidium Research Projects Within the Biohazard Assessment Research Branch 09/13/2007
VILLEGAS, E. Cryptosporidium Research Projects Within the Biohazard Assessment Research Branch. Presented at Workshop on Cryptosporidium Research Associated with Monitoring and Analytical Efforts in the Mid-Atlantic, Philadelphia, PA, September 13, 2007.
Abstract: Presented at the Workshop on Cryptosporidium Research Associated with Monitoring and Analytical Efforts in the Mid-Atlantic, September 13, 2007

PRESENTATION Biofilm-Like Water Sampling Device 09/05/2007
ASHBOLT, N. Biofilm-Like Water Sampling Device. Presented at 2007 Clean Water Summit, Cincinnati, OH, September 05 - 06, 2007.
Abstract: Presented at the 2007 Clean Water Summit, Cincinnati,OH, September 05-06, 2007

PRESENTATION Microarray for Detection of Waterborne Pathogens 09/05/2007
VILLEGAS, E. AND N. BRINKMAN. Microarray for Detection of Waterborne Pathogens. Presented at 2007 Clean Water Partnership Summit, Cincinnati, OH, September 05 - 06, 2007.
Abstract: To be presented at the 2007 Clean Water Partnership. September 5-6, 2007 at the USEPA, Cincinnati, OH

PRESENTATION Real-Time Microbial Ensor for Recreational Water 09/05/2007
DUFOUR, A. P. Real-Time Microbial Ensor for Recreational Water. Presented at 2007 Clean Water Summit, Cincinnati, OH, September 05, 2007.
Abstract: Presented at the 2007 Clean Water Summit, Cincinnati, OH. September 5-6,2007

PRESENTATION EPA's Expertise in Microbiological and Chemical Detection 09/05/2007
DUFOUR, A. P. EPA's Expertise in Microbiological and Chemical Detection. Presented at 2007 Clean Water Partnership Summit, Cincinnati, OH, September 05 - 06, 2007.
Abstract: To be presented at the 2007 Clean Water Partnership Summit held September 5-6, 2007 in Cincinnati, OH. The Challenge -Maintaining and Protecting Water Quality. Contaminants come from a variety of natural and man-made sources. Annually, 300 million tons of synthetic compounds derived from industrial and consumer products enter water systems.
Worldwide, over 2 million deaths each year can be attributed to biological contamination of water. In the wake of 9/11, there is heightened concern over the security of our nation's water infrastructure.

PRESENTATION Quantitative Pcr Analysis of Waterborne Legionella Species 09/05/2007
VESPER, S. J. AND R. A. HAUGLAND. Quantitative Pcr Analysis of Waterborne Legionella Species. Presented at 2007 Clean Water Summit, Cincinnati, OH, September 05 - 06, 2007.
Abstract: Presented at the 2007 Clean Water Summit held in Cincinnati, OH, September 5-6, 2007

PRESENTATION U.S. Environmental Protection Agency's Regulation and Management of Waterborne Viruses 08/27/2007
GRIMM, A. U.S. Environmental Protection Agency's Regulation and Management of Waterborne Viruses. Presented at International Symposium on Waterworks Technology, Seoul, SOUTH KOREA, August 27 - 28, 2007.
Abstract: The U.S. Environmental Protection Agency (USEPA) manages waterborne viruses and other pathogens through the establishment of rules and regulations that are designed to ensure public health protection. The rules that currently regulate pathogens focus on the management of viruses, Cryptosporidium, Giardia, Legionella and coliforms, and use a variety of approaches to reducing the level of these organisms in water. In addition, a Contaminant Candidate List (CCL) is maintained by the Agency as a way of identifying unregulated pathogens for which more data is needed before a regulatory determination can be made. Gathering more data on both regulated and unregulated pathogens is a major driver of the microbial research at the USEPA and each of the Laboratories and Centers that make up the Office of Research and Development (ORD) contribute to this effort by conducting complementary research that is organized according to the risk assessment paradigm.

PRESENTATION Dna Based Method of Mold and Applying the Environmental Relative Moldiness Index (Ermi) in Dod Facilities 08/07/2007
VESPER, S. J. Dna Based Method of Mold and Applying the Environmental Relative Moldiness Index (Ermi) in Dod Facilities. Presented at DoD Seminar - Force Health Protection Conference, Louisville, KY, August 07, 2007.
Abstract: To be presented at the Department of Defense - Force Health Protection Conference in Louisville, KY, August 7, 2007

PRESENTATION Understanding and Applying the Environmental Relative Moldiness Index (Ermi)SM in Dod Facilities 08/07/2007
VESPER, S. J. Understanding and Applying the Environmental Relative Moldiness Index (Ermi)SM in Dod Facilities. Presented at 2007 DoD 10th Annual Force Health Protection, Louisville, KY, August 07 - 10, 2007.
Abstract: Mold burdens in the indoor environment are a growing concern for the Department of Defense and other government agencies, as well as, the general public. Most recently mold in Walter Reed outpatient facilities became a significant issue. Yet there has been no standardized, objective method to quantify the mold burden indoors. This situation has now been corrected with the development of a DNA-based method of mold analysis called Mold Specific Quantitative Polymerase Chain Reaction (MSQPCR) (US Patent No.6,387,652). Using this technology to measure 36 indicator mold species in dust allowed for the development of a scale of relative mold burdens indoors called the Environmental Relative Moldiness Index (ERMI). This scale may be a useful tool to evaluate the mold levels in DOD facilities.

PRESENTATION Use of a Molecularly Imprinted Polymer in the Determination of Pyrethroid Pesticides in Composite Foods 07/22/2007
MORGAN, J. N., T. E. HIEBER, A. M. PAWLECKI-VONDERHEIDE, P. KAUFFMAN, L. J. MELNYK, B. BOYD, A. RYBERG, AND E. YLMAZ. Use of a Molecularly Imprinted Polymer in the Determination of Pyrethroid Pesticides in Composite Foods. Presented at 2007 Florida Pesticide Residue Workshop, St. Pete Beach, FL, July 22 - 25, 2007.
Abstract: The U.S. Environmental Protection Agency's (EPA) National Exposure Research Laboratory (NERL) measures the exposure of individuals to chemical pollutants through the diet, as well as other media. In support of this research, methods are being evaluated for determination of various classes of pesticides in composite diet samples. Existing methods for pesticides generally have been developed for regulatory purposes and often do not have sufficiently low detection limits for exposure studies. Consequently, there is a need to improve the performance of these methods for analysis of pesticides in composite dietary samples. The objective of this work is to evaluate the applicability of a molecularly imprinted polymer (MIP) based solid phase extraction material for use in the analysis of pyrethroid pesticides in composite dietary samples collected in exposure studies.
A MIP-based solid phase extraction material, specific for pyrethroid pesticides, was developed by MIP Technologies AB, under contract to the EPA. This material was evaluated in combination with a variety of extraction and clean-up techniques in an effort to optimize its performance in the determination of pyrethroid pesticides in composite foods. This presentation will outline the various experiments conducted until acceptable performance was achieved.

The final method consisted of a modified QuEChERS1 procedure followed by a clean-up step using the MIP. Detection was performed using GC/µECD. The procedure was evaluated for cis and trans-permethrin, cypermethrin and cyfluthrin in high, medium and low fat (10%, 5% and 2%, respectively) food samples. Recoveries ranged from 83-126%. Detection limits ranged from 7 to 35 ppb using GC/ECD. This work was designed primarily to evaluate performance of the MIP. Future work will focus on ways to improve detection limits to sub-ppb levels.

PRESENTATION Emerging Contaminants in the Water Cycle: Fate and Transport 07/16/2007
GLASSMEYER, S. Emerging Contaminants in the Water Cycle: Fate and Transport. Presented at USEPA Workshop on Building an Integrated Surveillance System for Emerging Chemicals in the Great Lakes and Nationwide, Chicago, IL, July 16 - 18, 2007.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the complex mixtures of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. In the source water of one conventional drinking water facility, 45 out of 113 monitored chemicals were detected at least once, with 21 chemicals still detectable in the finished drinking water. This documents the incomplete removal of such chemicals during treatment. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Dna Based Method of Mold and Applying the Environmental Relative Moldiness Index (Ermi) 07/12/2007
VESPER, S. J. Dna Based Method of Mold and Applying the Environmental Relative Moldiness Index (Ermi). Presented at NASA Seminar, Cincinnati, OH, July 12, 2007.
Abstract: NASA facilities can potentially have mold contamination problems. The EPA has created an Environmental Relative Moldiness Index based on the analysis of dust by Mold Specific Quantitative PCR (MSQPCR). In this presentation, the scientific background for the ERMI will be presented. The goal of the ERMI is to be able to provide a simple, comparative value that will define the mold burden in an indoor environment.

PRESENTATION Development of An EPA Method for Perfluorocalkyl Compounds in Drinking Water 06/25/2007
SHOEMAKER, J. A. Development of An EPA Method for Perfluorocalkyl Compounds in Drinking Water. Presented at 2007 American Water Resources Association, Vail, CO, June 25 - 27, 2007.
Abstract: Perfluoroalkyl compounds (PFCs) have been manufactured for over 50 years and their use has dramatically increased over the years. Due to their unique properties of repelling both water and oil, PFCs have been used in a wide variety of applications. In 2001, identification of organic fluorine as PFCs was confirmed in human sera through the use of liquid chromatography/mass spectrometry (LC/MS). These compounds received world-wide attention when the presence of perfluorooctane sulfonate and perfluorooctanoic acid were reported in blood and liver samples of animals in urban and remote locations.
Of particular interest is the global detection of PFCs in ground and surface waters as these can be potential drinking water sources. The 1996 Amendments to the Safe Drinking Water Act required the Environmental Protection Agency (EPA) to establish a Drinking Water Contaminant Candidate List (CCL) that contains a list of drinking water contaminants that the Agency will consider for future regulation. Because of the recent interest in PFCs, it is likely that they will be listed on a future CCL. One of the key pieces of information necessary to make a regulatory determination is nationwide occurrence data for the chemical contaminants under consideration. Historically, EPA's Office of Ground Water and Drinking Water has collected the necessary occurrence data under its Unregulated Contaminant Monitoring Regulations. To gather occurrence data, a rugged analytical method is needed for these PFCs.

While several methods have been reported, these methods do not adequately address issues specific to analyzing PFCs in drinking water. Issues, such as preservatives, internal and surrogate standards, and establishing acceptable background levels, will be studied in this method development effort. The target analyte list includes the C6 through C14 perfluorinated carboxylic and sulfonic acids, as well as the perfluorooctanesulfonamidoacetates. Drinking water samples are concentrated by solid phase extraction using styrene-divinyl benzene sorbents and analyzed using LC/tandem mass spectrometry (LC/MS/MS). Recovery and precision data for the target PFCs will be presented using various drinking water matrices. Detection limits below 10 ng/L will be demonstrated.


PRESENTATION Emerging Contaminants in the Drinking Water Cycle 06/25/2007
GLASSMEYER, S., P. E. STACKELBERG, E. T. FURLONG, AND D. W. KOLPIN. Emerging Contaminants in the Drinking Water Cycle. Presented at 2007 AWRA Summer Specialty Conference, Vail, CO, June 25 - 27, 2007.
Abstract: PRESENTATION OUTLINE: I. General overview of the water cycle;
II. USEPA and USGS Research;

a. Wastewater treatment plant (WWTP) effluents and downstream surface waters;

b. Groundwater down gradient from WW lagoon;

c. Source and finished water from a drinking water treatment plant (DWTP);

d. Chlorination laboratory studies;

III. Future directions


PRESENTATION Global Water Research Coalition 06/25/2007
GLASSMEYER, S., F. S. HAUCHMAN, AND D. TILLMAN. Global Water Research Coalition. Presented at 2007 AWRA Summer Specialty Conference, Vail, CO, June 25 - 27, 2007.
Abstract: The Global Water Research Coalition (GWRC) is a collaboration of 14 member drinking and wastewater research organizations. The USEPA is currently a partner to the GWRC membership. Through the GWRC, the members are able to leverage research funds on mutually desired efforts to maximize outcomes and minimize duplication of efforts. Currently, these research efforts cover topics such as algal toxins, endocrine disrupting chemicals, pharmaceuticals and personal care products, nitrosodimethylamine, water quality in distribution systems, asset management and water reuse.
Issues that cannot be adequately addressed by individual organizations

Exchange of knowledge and expertise needed

PRESENTATION Development and Evaluation of a Microarray Approach to Detect and Genotype Noroviruses in Water 06/18/2007
BRINKMAN, N. Development and Evaluation of a Microarray Approach to Detect and Genotype Noroviruses in Water. Presented at The U.S. EPA Workshop on Innovative Approach to Detecting Microorganisms in Water, Cincinnati, OH, June 18 - 20, 2007.
Abstract: Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. These viruses are usually detected and genotyped using reverse transcription-polymerase chain reaction (RT-PCR) based methods followed by sequencing. Unfortunately, the accurate detection of noroviruses in environmental samples is often hindered by the co-amplification of non-specific DNA, which can result in the need for further purification of PCR products before accurate sequence information can be obtained. As an alternative to direct sequencing, a generic microarray was evaluated for its ability to genotype norovirus RT-PCR products by probe hybridization. With this approach, RT-PCR amplicons were first mixed with a range of genotype specific probes and single base extension (SBE) reactions were run. This resulted in the labeling of those probes that have sequences complementary to specific RT-PCR products. These genotype-specific probes were then hybridized to an Affymetrix GenFlex Tag Array for detection. Using a standardized, multiplex SBE reaction, the genotyping of representative strains was accomplished and resulted in the generation of specific hybridization patterns, or fingerprints, on the microarray that were diagnostic for the genotype of norovirus detected. Furthermore, the SBE-GenFlex array method was shown to be successful in the genotype identification of noroviruses seeded into tap and Ohio River water samples. This study demonstrates the utility of using a microarray to genotype noroviruses in complex environmental matrices.

PRESENTATION An Overview of Pathogen Research in the Microbiological and Chemical Exposure Assessment Research Division 06/18/2007
GRIMM, A. An Overview of Pathogen Research in the Microbiological and Chemical Exposure Assessment Research Division. Presented at USEPA Workshop on Innovative Approaches for Detecting Microorganisms in Water, Cincinnati, OH, June 18 - 20, 2007.
Abstract: The Microbiological and Chemical Exposure Assessment Research Division of the EPA Office of Research and Development's National Exposure Research Laboratory has a robust in-house research program aimed at developing better occurrence and exposure methods for waterborne pathogens. In particular, research is aimed toward developing and improving occurrence methods so that they are more rapid, sensitive and inexpensive. To conduct this research, a diverse array of detection technologies is used, including real-time PCR, microarrays, proteomics, and cell culture, among others. In addition, the division has more recently invested in evaluating new approaches to collecting and concentrating samples more effectively. The long-term goal of this work is to enable the Agency to conduct better, more accurate risk assessments that will aid regulatory decision-making.

PRESENTATION Decision-Making and Needs for Support: Protecting Groundwater Supplies from Pathogen Health Risks International Perspective 06/12/2007
ASHBOLT, N. Decision-Making and Needs for Support: Protecting Groundwater Supplies from Pathogen Health Risks International Perspective. Presented at Pathogen-In-Ground Research Consortium - Kick Off Workshop - Canadian Water Network, Toronto, ON, CANADA, June 12 - 13, 2007.
Abstract: To be presented at the Pathogen-In-Ground Research Consortium - Kick Off Workshop - Canadian Water Network, Toronto, Canada, June 12-13, 2007

PRESENTATION Laboratory Analyses: Water and Environmental Samples 06/01/2007
FOUT, G. Laboratory Analyses: Water and Environmental Samples. Presented at Workshop for Improving the Recognition, Investigation, and Reporting of Waterborne Disease Outbreaks Associated with Drinking, Recreational and Other Waters, Nashville, TN, May 27 - June 01, 2007.
Abstract: To be presented at the Workshop for Improving the Recognition, Investigation, and Reporting of Waterborne Disease Outbreaks Associated with Drinking, Recreational and Other Waters in Nashville, TN, May 29 - June 1, 2007

PRESENTATION Presentations at California Water Environment Association Specialty Conference on Chemicals of Emerging Concern 05/22/2007
GLASSMEYER, S., E. T. FURLONG, AND D. KOLPIN. Presentations at California Water Environment Association Specialty Conference on Chemicals of Emerging Concern. Presented at California Water Environment Association Specialty Conference on Chemicals of Emerging Concern, Van Nuys, CA, May 22 - 24, 2007.
Abstract: 1. Introduction to Emerging Contaminants
2. Why are emerging Contaminants Important?

3. Regulatory Response to Emerging Contaminants

4. Identifying Chemical Compounds from Wastewater Discharge

PRESENTATION Real-Time Quantitative Pcr Detection of Mycobacterium Avium Complex Organisms in Drinking Water 05/21/2007
KING, D. N., A. BEUMER, AND S. L. PFALLER. Real-Time Quantitative Pcr Detection of Mycobacterium Avium Complex Organisms in Drinking Water. Presented at American Society for Microbiology 107th General Meeting, Toronto, ON, CANADA, May 21 - 25, 2007.
Abstract: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U.S. Environmental Protection Agency's Contaminant Candidate List (CCL) due to their association with human disease and occurrence in public drinking water systems. Current methods for detecting MAC organisms in drinking water are culture-based. However evidence suggests that culture-based methods have severe limitations including long incubation periods, loss of target due to overgrowth of background organisms, up to 70% loss of target due to harsh decontamination techniques, and inability to recover MAC in a viable-but-non-culturable state. Because of these drawbacks and the need for more accurate and comprehensive occurrence data, we have developed real-time QPCR assays for the detection and quantification of MA, MI, and M. avium subspecies paratuberculosis (MAP) in drinking water. Real-time QPCR assays were developed using primers and TaqMan® probes designed to amplify a region of the 16S rDNA in MA and MI, and regions of IS900 and Target 251 in MAP. Primer/probe sets were found to be highly specific when compared to
sequences in nucleotide databases and confirmed experimentally by screening 104 MAC strains. No false negatives occurred when each species was tested with its own primer/probe set, 2.3% (1/42) MA strains were false positive with the MI prober/probe set, and 2.5% (1/40) MI strains were false positive with the MA primer/probe set. No false positives were obtained when nine non-MAC species were screened with all primer/probe sets. Quantification is linear over a minimum range of six logs of target

concentration in all four assays. Additionally, a control has been developed to measure PCR inhibition due to compounds in the water matrix. We are currently evaluating the QPCR assays for use on actual drinking water samples as a rapid alternative to culture methods in order to generate a more complete understanding of MAC occurrence in drinking water.

PRESENTATION Examination of the Protein Profile of Helicobacter Pylori Under Different Growth Conditions Using Matrix-Assisted Laser Desorption Mass Spectrometry 05/21/2007
STELMA, G. N., D. J. FLANIGAN, L. A. BOCZEK, D. J. LYE, AND M. J. DONOHUE. Examination of the Protein Profile of Helicobacter Pylori Under Different Growth Conditions Using Matrix-Assisted Laser Desorption Mass Spectrometry. Presented at American Society for Microbiology 107th Meeting, Toronto, ON, CANADA, May 21 - 25, 2007.
Abstract: US EPA currently has H. pylori on its Contaminant Candidate List 2 (CCL 2), methods are needed to detect the occurrence of viable H. pylori in drinking water. H. pyloi is an interesting microorganism because it can change from a cultural and metabolically active state with a helical morphology to a non-culturable, dormant state with a coccoid morphology. There is currently no culture medium for isolating H. pyloi from drinking water. Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry
(MALDI-MS) was used to look at protein profiles of H. pyloi over the course of 10 days. After four days on blood agar, more than 50% of the H. pyloi cells were coccoid and non-culturable and a noticeable shift of protein expression was observed. This altered protein profile was continuously observed up to day 10, at which time all culturability was lost but ATP was still present. Efforts are currently focused on identifying the H. pyloi proteins observed between day 4 and day 10. We hope to use these proteins as possible markers of viability and develop more sensitive detection methods using these markers as targets.

PRESENTATION Efficient Recovery of Enterococci from Marine and Fresh Water Beaches By a 30,000 Molecular Weight Cutoff Hollow Fiber Ultrafilter 05/21/2007
MCDANIELS, A. E., C. C. RANKIN, L. J. WYMER, A. P. DUFOUR, AND K. OSHIMA. Efficient Recovery of Enterococci from Marine and Fresh Water Beaches By a 30,000 Molecular Weight Cutoff Hollow Fiber Ultrafilter. Presented at American Society for Microbiology General 107th Meeing, Toronto, ON, CANADA, May 21 - 25, 2007.
Abstract: Ultrafiltration systems have been used to concentrate pathogens from various types of fresh water samples. However, less work has been done with marine waters for the concentration of pathogens or indicator bacteria. An ultrafiltration approach to concentrate indicator bacteria such as Enterococci may be advantageous because of the ability to smaple larger volumes and concentrate multiple target organisms simultaneously. Each ten liter sample was seeded with approximately 1 x 104 cells. A peristaltic pump was used to force water through a 30,000 molecular weight cutoff hollow fiber ultrafilter (Fresenius). During the filtration process, Enterococci remained in the retentate while the filtered water was collected in the permeate. Cells were eluted from the filter by forward flushing with 0.1% Tween 80 and 0.05M glycine in PBS at 7.2 pH, followed by reverse flushing and filter shaking. Nitrogen gas was used to remove all remaining fluid. Ten liters were reduced to approximately 500 mL at a rate of l liter per minute. Aliquots from seeded initial and final concentrates were filtered, the filtgers placed on mEI agar for growth and colony counts used to calculate filter mL at a rate of 1 liter per minute. Aliquots from seeded initial and final concentrates were filtered, the filters placed on mEI agar for grown and colony counts used to calculate recoveries. Recoveries from seeded marine waters averaged 93% (n=17) from the Pacific and 64.7 (n=13) from the Atlantic Oceans, which was a significant difference (p-value=0.0185). Seeded fresh water recoveries averaged 81% (n=12). The physical-chemcial parameters of pH, turbidity, specific conductivity and salinity were within their expected ranges. In conclusion, the ultrafiltration process produced high recoveries of Enterococci from large volumes of beach waters within a reasonable time frame, was simple to operate, used inexpensive disposable filters and can be operated in the field as well as in the laboratory.

PRESENTATION Sample Preparation for Metallomics Studies 05/21/2007
PAWLECKI-VONDERHEIDE, A. M. Sample Preparation for Metallomics Studies. Presented at CERMACS 2007, Covington, KY, May 21, 2007.
Abstract: To be presented at the CERMACS 2007 - Central Regional Meeting of the American Chemical Society, Northern Kentucky Convention Center, Covington, KY May 21, 2007

PRESENTATION Dietary Arsenic Exposure Assessment Using Enzymatic Based Extraction Conditions and Detection of Urinary Thio-Arsenicals as Metabolites of Exposure Mceard2 05/21/2007
CREED, J. T., P. A. CREED, J. ELLIS, S. CONKLIN, C. GALLAWA, A. YOUNG, C. A. SCHWEGEL, AND J. CARUSO. Dietary Arsenic Exposure Assessment Using Enzymatic Based Extraction Conditions and Detection of Urinary Thio-Arsenicals as Metabolites of Exposure Mceard2. Presented at Central Regional Meeting of the American Chemical Society (CERMACS), Covington, KY, May 21, 2007.
Abstract: Inorganic arsenic is classified as a carcinogen and has been linked to lung and bladder cancer as well as other non-cancerous health effects. Because of these health effects the U.S. EPA has set a Maximum Contaminant Level (MCL) at 10ppb based on a linear extrapolation of risk and a Health Risk Reduction and Cost Analysis (HRRCA). A re-evaluation of the HRRCA will require the Agency to review scientific developments regarding arsenic's Mode of Action, treatment options and the aggregate exposure assessment. The scientific basis for any drinking water MCL is reviewed every six years.

PRESENTATION Temporal Variability of Microbial Indicators of Fecal Contamination of Marine and Freshwater Beaches 05/21/2007
WYMER, L. J., A. P. DUFOUR, AND C. MCGEE. Temporal Variability of Microbial Indicators of Fecal Contamination of Marine and Freshwater Beaches. Presented at American Society for Microbiology General 107th Meeting, Toronto, BC, CANADA, May 21 - 25, 2007.
Abstract: Monitoring methods for microbial indicators of fecal contamination are an integral component for protecting the health of swimmers exposed to potentially contaminated bathing beach waters. The design of monitoring systems which will accurately characterize the quality of water is dependent on knowledge of the variability associated with water sampling. This study was conducted to determine the
temporal variability encountered at bathing beaches. High frequency sampling was conducted at marine and freshwater beaches over a sixty day period. Multiple samples were taken at minute, 10 minute, hourly and daily time intervals. Water samples were assayed for E. coli or enterococci using membrane filter methods. In general, as the time interval between samples increased so did their variability. Based on the average freshwater and marine results, samples taken within seconds of each other had a coefficient of variation (CV) of 0.7. Samples taken at 10 minute intervals had a CV of 1.1. Samples taken at hourly intervals had a CV 1.7, and samples taken daily had a CV of 2.2. The results of this study show that single samples do not adequately characterize the quality of beach waters and that temporal variability must be given serious consideration when developing sampling plans for beach waters.

PRESENTATION Children's Exposure to Pesticides: Unconventional Routes of Introduction 05/18/2007
PAWLECKI-VONDERHEIDE, A. M., L. J. MELNYK, J. N. MORGAN, T. E. HIEBER, AND P. KAUFFMAN. Children's Exposure to Pesticides: Unconventional Routes of Introduction. Presented at Seminar for the University of Cincinnati, College of Engineering, Cincinnati, OH, May 18, 2007.
Abstract: To be presented at Seminar for the University of Cincinnati, College of Engineering, May 18, 2007

PRESENTATION A Faster Method of Measuring Recreational Water Quality 05/14/2007
HAUGLAND, R. A. A Faster Method of Measuring Recreational Water Quality. Presented at EPA Region 10 Bacteria Conference, Tacoma, WA, May 14 - 15, 2007.
Abstract: To be presented at the EPA Region 10 Bacteria Conference, Tacoma, Washington, May 14-15, 2007

PRESENTATION Urban Water System Pathogen Assessment: Significance of Distribution Biofilms 05/10/2007
ASHBOLT, N. Urban Water System Pathogen Assessment: Significance of Distribution Biofilms. Presented at Center for Disease Control Meeting, Atlanta, GA, May 10, 2007.
Abstract: Quantitative microbial risk assessment (QMRA), while not new to science is now providing a fundamental role in framing water guidelines internationally as well as identifying research gaps to be filled. Professor Ashbolt has been instrumental in working QMRA concepts into WHO guidelines for drinking, recreational and reuse waters, as well as within the Swedish Urban Water program and a recently completed EU project (MicroRisk). The process steps in undertaking a QMRA will be discussed along with Bayesian statistical methods that are appropriate to handle small pathogen datasets from watersheds through water treatment. Finally, the role of biofilms in potentially enhancing pathogen risks (through pathogen accumulation and in some cases growth) will be discussed, for both drinking and recycled water applications.

PRESENTATION Analysis of Water Soluble Volatile Organic Chemicals in Drinking Water When Volatiles Aren't Purgeable 05/10/2007
MUNCH, J. W. AND P. GRIMMETT. Analysis of Water Soluble Volatile Organic Chemicals in Drinking Water When Volatiles Aren't Purgeable. Presented at 45th Annual Water Workshop, Columbus, OH, May 10, 2007.
Abstract: Slide and poster to be presented at the 45th Annual Water Workshop in Columbus, OH, May 10, 2007

PRESENTATION Urban Water System Pathogen Assessments: Significance of Distribution Biofilms 05/04/2007
ASHBOLT, N. Urban Water System Pathogen Assessments: Significance of Distribution Biofilms. Presented at Urban Water System Pathogen Risk Assessments: Signficance of distribution of biofilms, Cincinnati, OH, May 04, 2007.
Abstract: Quantitative microbial risk assessment (QMRA), while not new to science is now providing a fundamental role in framing water guidelines internationally as well as identifying research gaps to be filled. Professor Ashbolt has been instrumental in working QMRA concepts into WHO guidelines for drinking, recreational and reuse waters, as well as within the Swedish Urban Water program and a recently completed EU project (MicroRisk). The process steps in undertaking a QMRA will be discussed along with Bayesian statistical methods that are appropriate to handle small pathogen datasets from watersheds through water treatment. Finally, the role of biofilms in potentially enhancing pathogen risks (through pathogen accumulation and in some cases growth) will be discussed, for both drinking and recycled water applications.

PRESENTATION Cryptosporidium Source Tracking to Enhance Source Water Protection Implementation in the Potomac River Watershed: A Regional Applied Research Efforts (Rare) Project 04/30/2007
YANG, W., P. CHEN, R. B. LANDY, C. KANETSKY, E. VILLEGAS, AND L. XIAO. Cryptosporidium Source Tracking to Enhance Source Water Protection Implementation in the Potomac River Watershed: A Regional Applied Research Efforts (Rare) Project. Presented at U.S. EPA Region 5 Laboratory Technical Information Group Conference, Chicago, IL, April 30 - May 03, 2007.
Abstract: The Potomac River watershed is a critical drinking water supply for the Washington DC metropolitan area. In 2004, the Drinking Water Source Protection Partnership (DWSPP) was formed to help coordinate efforts by local drinking water utilities and government agencies to protect this watershed from various contaminants, including Cryptosporidium. This organism is a protozoan parasite that is excreted in feces by infected animals and humans. There are many different species of Cryptosporidium, but only two, C. parvum and C. hominis, are commonly associated with human infection. Although the current standard method used to detect Cryptosporidium in water is effective in enumerating oocysts, it cannot differentiate human from animal forms of Cryptosporidium. This limitation thus prevents the identification of specific Cryptosporidium species/genotypes that are contaminating environmental waters. Recently, a nested-PCR-based genotyping method has been developed and used to detect and identify specific Cryptosporidium species/genotypes in the watershed. Since Cryptosporidium is host specific, this technique has also been useful in tracking potential sources of Cryptosporidium contamination within the watershed. For this RARE project, a nested-PCR- method is used to detect and identify various Cryptosporidium genotypes that are present and to determine their likely sources in the Potomac River watershed. Results from this project will help DWSPP and local drinking water utilities evaluate current management practices that are aimed at minimizing Cryptosporidium contamination of their watershed.

PRESENTATION Estimating Pathogen Exposures the Critical Challenge for Qmra to Support Regulation and Management of Waters 04/23/2007
ASHBOLT, N. Estimating Pathogen Exposures the Critical Challenge for Qmra to Support Regulation and Management of Waters. Presented at Toxicology and Risk Assessment Conference, West Chester, OH, April 23 - 26, 2007.
Abstract: Pathogen and indicator concentrations normally vary by several orders of magnitude in raw waters, and to an even greater extent during hazardous event periods. This variation in concentration typically dominate the estimate of infection generated in a quantitative microbial risk assessment (QMRA), particularly if results are not averaged over a year. In addition to this variation, numerous uncertainties result from our attempts to assay pathogens and model their behaviour through water systems.
Raw water [oo]cyst concentrations are typically presented with little or no reporting of specific recovery data representative of the site sampled. The uncertainty resulting from limited recovery data was estimated for the MicroRisk Project systems using available data to improve the quality of Cryptosporidium and Giardia raw water concentration estimates. Recovery datasets ranging from 3-99 data points were examined by Bayesian statistics representing three Approaches: I - no recovery data, II - limited, unpaired recovery data from samples, and III - paired recovery data. No useful relationships were seen between water turbidity or type and recoveries reported. Critically, Approach I underestimated [oo]cyst concentrations by about 100%, with little difference between Approaches II & III. Using the smallest (n=3) recovery dataset, the upper band of uncertainty were on average more than 10-times (and on occasion up to 100-times) greater than when using the fullest (n=99) dataset; however, limited reduction in uncertainty occurred beyond n = 20. Nonetheless, for QMRA purposes, recovery data should be collected as a pair with count data for an initial period at least, so that any relationships (priors in Bayesian statistics) may be ascertained without the need to rely on and apply trends from elsewhere. When conveying QMRA results for risk management, output uncertainty should be incorporated into the results via either a two-dimensional (variability and uncertainty) risk assessment or a sensitivity analysis that includes recovery uncertainties.


PRESENTATION Internal Amplification Control for Use in Quantitative Polymerase Chain Reaction Fecal Indicator Bacteria Assays 04/13/2007
SIEFRING, S., E. ATIKOVIC, R. A. HAUGLAND, M. SIVAGANESAN, AND O. C. SHANKS. Internal Amplification Control for Use in Quantitative Polymerase Chain Reaction Fecal Indicator Bacteria Assays. Presented at Ohio Branch of the American Society for Microbiology Annual Meeting, Canton, OH, April 13 - 14, 2007.
Abstract: Quantitative polymerase chain reaction (QPCR) can be used as a rapid method for detecting fecal indicator bacteria. Because false negative results can be caused by PCR inhibitors that co-extract with the DNA samples, an internal amplification control (IAC) should be run with each sample. Currently available controls used in QPCR analyses for the fecal indicator bacterial groups Enterococcus and Bacteroidetes were designed primarily to determine variability in DNA yields from environmental samples and cannot be used to directly demonstrate PCR inhibition. Therefore, a competitive IAC plasmid DNA was constructed to detect the presence of PCR inhibitors in QPCR assays for both Enterococcus and Bacteroidetes rRNA gene targets. The IAC was designed to contain a single site for hybridization with a unique probe sequence that is flanked by multiple primer-hybridizing sites that corresponded to the same primers used in the Enterococcus, Bacteroidetes and several additional QPCR assays. The IAC construct was prepared by overlap extension PCR, inserted into the pCR4®TOPO plasmid vector (Invitrogen) and cloned. Gel electrophoresis, QPCR and sequencing analyses were performed to confirm the presence of the correct IAC sequences in the plasmid. Slope and intercept values of standard curves generated from genomic DNA in simplex analyses were not significantly different (p > 0.05) from the values generated during multiplex analyses with a fixed number of 25 IAC plasmid copies. Ranges of genomic DNA concentrations that did not significantly affect the IAC results under the same conditions were also established. Multiplex analyses with the IAC were used in a study of the relative levels of Enterococcus and Bacteroidetes DNA in fecal samples from cattle. In these analyses the Enterococcus IAC assay results showed highly consistent cycle threshold values (mean = 34.15, std. deviation = 0.69, N = 159) where only three results failed to occur within the 95% confidence interval established from analyses of control samples with IAC plasmid but no fecal extracts present. Greater variability in the Bacteroidetes IAC assay results was consistent with the relatively high levels of genomic DNA from these organisms in the samples. These studies indicate that the IAC plasmid DNA performs well as an inhibition control and also may be useful as an alternative to genomic DNA standards for quantifying fecal bacteria target DNA sequences.

PRESENTATION Understanding and Applying Environmental Relative Moldiness Index Ermi 04/03/2007
VESPER, S. J. Understanding and Applying Environmental Relative Moldiness Index Ermi. Presented at National EPA-Tribal Science Council Meeting, Chicago, IL, April 03, 2007.
Abstract: This study compared two binary classification methods to evaluate the mold condition in 271 homes of infants, 144 of which later developed symptoms of respiratory illness. A method using on-site visual mold inspection was compared to another method using a quantitative index of moldiness, calculated from mold specific quantitative PCR (MSQPCR) measurements on the concentration of 36 species of molds in floor dust samples called the EPA relative moldiness index© (ERMI©).
The binary classification of homes as either moldy or non-moldy by on-site visual home inspection was not predictive of the development of respiratory illness (wheeze and/or rhinitis) (p = 0.27). Conversely, a method developed using the ERMI© index fit to a logistic function, can be used to predict the occurrence of illness in homes and allows stake-holders the choice among various levels of risk. An example is given where an ERMI© value of -4.29 is used as a threshold for binary classification of homes producing an odds ratio of 2.53 (95% confidence limits of 1.35, 4.77). The ERMI© based method provides a new and more flexible platform to support mold remediation decisions based on quantitative estimates using cost and risk tolerance.


PRESENTATION Ultraviolet-and Solar Light-Activated Nanostructured Tio2 Photocatalysts: Application in the Destruction of Cyanotoxins, a Group of Cyanotoxins Emerging Drinking Water Contaminants 03/29/2007
DIONYSIOU, D. D., M. G. ANTONIOU, H. CHOI, A. A. DELACRUZ, AND J. A. SHOEMAKER. Ultraviolet-and Solar Light-Activated Nanostructured Tio2 Photocatalysts: Application in the Destruction of Cyanotoxins, a Group of Cyanotoxins Emerging Drinking Water Contaminants. Presented at American Chemical Society National Meeting, Chicago, IL, March 25 - 29, 2007.
Abstract: To be presented at the American Chemical Society National Meeting in Chicago, IL, March 25-29, 2007

PRESENTATION Comparison of the Urinary Metabolites of Rats, Mice, and Humans After Oral Arsenic Exposure Focusing on Thioarsenicals 03/25/2007
CONKLIN, S., B. ADAIR, P. A. CREED, J. T. CREED, M. F. HUGHES, AND D. J. THOMAS. Comparison of the Urinary Metabolites of Rats, Mice, and Humans After Oral Arsenic Exposure Focusing on Thioarsenicals. Presented at American Chemical Society Meeting, Chicago, IL, March 25 - 29, 2007.
Abstract: Urinary metabolites of arsenic are useful as biomarkers of exposure because ingested arsenic is excreted primarily in urine1. Complete urinary arsenic speciation can provide insight into possible metabolic pathways as well as potential exposure sources. The pattern of excreted metabolites depends upon which arsenicals are ingested. For example, both arsenosugars and inorganic arsenic generate dimethylarsinate (DMA) as a major urinary metabolite. For arsenosugars, this involves cleavage of As-C bonds, while for inorganic arsenic, As-C bonds are formed. In comparison, other arsenicals such as arsenobetaine and arsenocholine are excreted unchanged in urine. The recent finding of sulfur analogs of some arsenic oxides indicates that comprehensive urinary speciation has not yet been achieved. These sulfur analogs pose a unique analytical challenge because of the possible solution-phase interconversion between the oxygen and sulfur containing metabolites. The observed distribution between the two forms may be influenced by sample preparation and handling because of this equilibrium. It is with this in mind that a series of urine samples from rats, mice and humans have been analyzed specifically for the thiolated arsenicals dimethylthioarsinic acid (DMTA) and trimethylarsine sulfide (TMAS). The results are compiled here as an overview of the thio-arsenicals detected in urine (other arsenicals such as DMA and arsenate (As(V)) were detected, but are not included in this presentation).

PRESENTATION Sulfate Radical-Based Advanced Oxidation Processes 03/25/2007
DIONYSIOU, D. D., A. RASTOGI, M. G. ANTONIOU, G. P. ANIPSITAKIS, S. R. AL-ABED, A. A. DELACRUZ, AND J. A. SHOEMAKER. Sulfate Radical-Based Advanced Oxidation Processes. Presented at American Chemical Society National Meeting, Chicago, IL, March 25 - 29, 2007.
Abstract: This will be presented at the American Chemical Society National Meeting, Chicago, IL, March 25-29, 2007

PRESENTATION Global Warming and Trans-Boundary Movement of Waterborne Microbial Pathogens 02/26/2007
ASHBOLT, N. Global Warming and Trans-Boundary Movement of Waterborne Microbial Pathogens. Presented at International Symposium on Dialogue between Social and Natural Sciences, Honolulu, HI, February 26 - 28, 2007.
Abstract: Subtle increases in temperatures can have profound impacts on the prevalence of various waterborne microbial pathogens. Such impacts may be seen in three major areas: 1) fecally-contaminated drinking waters; 2) fresh produce that has been irrigated or processed with contaminated water; and 3) seafood where pathogens and microbial toxins are present. Each of these areas is influenced by rainfall events, which have a fundamental influence on the fate and transport of pathogens.
Temperature alone can also impact, as seen with the epidemic strains of cholera (disease from certain Vibrio cholerae bacteria). In coastal environments, cholera outbreaks associate with particular phytoplankton blooms in nutrient-enriched coastal waters; with blooms varying due to changing precipitation regimes and El Niño. A further ecological interaction is seen by bacteriophage mediated cholera toxin genes inserted into non-toxic V. cholerae strains.

For microorganisms, boundaries occur at the tens of micron scale, with most terrestrial microbes living in slime on surfaces (biofilm). As ambient water temperatures increase, there is a clear increase in the growth of biofilms and their associated pathogens, many of which appear to be amoeba-associated. Most opportunistic bacterial pathogens (such as strains of Aeromonas, Legionella, Mycobacterium) and even frank, fecally-derived pathogens (e.g., Campylobacter jejuni, Cryptosporidium parvum, and enteric viruses) have been observed to accumulate within biofilm amoeba. With rising temperatures and increased application of recycled waters, biofilm pathogens will presenting a range of pathogen challenges to communities.

Human behavior is also influenced by climate change, with increased consumption of uncooked foods and water during warmer conditions, as well as increased travel (trans-boundary transport of pathogens). Increased rainfall events also promote zoonotic diseases (e.g., cryptosporidiosis, E. coli O157:H7 infections) via contamination of drinking water sources and waters used to irrigate crops.


PRESENTATION A Study of the Interconversion of Methylated Arsenic Oxides to Methylated Arsenic Sulfides in Solutions Containing Free Sulfide 02/18/2007
CONKLIN, S., P. A. CREED, AND J. T. CREED. A Study of the Interconversion of Methylated Arsenic Oxides to Methylated Arsenic Sulfides in Solutions Containing Free Sulfide. Presented at 2007 European Winter Conference on Plasma Spectrochemistry, Taormina, ITALY, February 18 - 23, 2007.
Abstract: Evidence suggests that thiolated arsenicals are urinary metabolites in both humans and rats. These thiolated species may be formed in the digestive system or as metabolites within the body. The role they may play in the overall toxicity of arsenic is an active area of research. This research effort would benefit from an improved understanding of how the oxide and thiolated forms of arsenic can interconvert based on matrix constituents.
Data will be presented demonstrating that trimethylarsine oxide (TMAO), dimethlyarsinic acid (DMA) and monomethylarsonic acid (MMA) all produce thiolated analogs in the presence of solution phase sulfide. This conversion is shown to be pH sensitive and the conversion rate is shown to increase in the following order: MMA

PRESENTATION Recreational Water Quality and Swimming Associated Health Effects 7th Annual Water Monitoring Conference 02/01/2007
DUFOUR, A. P. Recreational Water Quality and Swimming Associated Health Effects 7th Annual Water Monitoring Conference. Presented at 7th Annual Water Monitoring Conference, Ames, IA, February 01 - 02, 2007.
Abstract: To be presented at the 7th Annual Water Monitoring Conference, Ames, Iowa, February 1-2, 2007

PRESENTATION Use of Data to Inform Characterization and Management in Addressing Biofilm Problems 01/31/2007
ASHBOLT, N. Use of Data to Inform Characterization and Management in Addressing Biofilm Problems. Presented at TCR Stakeholder Workshop, Washington, DC, January 31 - February 01, 2007.
Abstract: To be presented at the TCR Stakeholder Workshop, Washington, DC, January 31-February 1, 2007

PRESENTATION Collection of Water Samples 01/09/2007
BRENNER, K. P. Collection of Water Samples. Presented at UPRM Water Microbiology Methods Course, Mayaguez, PR, January 09 - 12, 2007.
Abstract: Powerpoint presentation to be presented at the UPRM Water Microbiology Methods Course, Mayaguez, Puerto Rico, January 9-12, 2007

PRESENTATION Water Microbiology Methods: Membrane Filter Techniques 01/09/2007
BRENNER, K. P. Water Microbiology Methods: Membrane Filter Techniques. Presented at UPRM Water Microbiology Methods Course, Mayaguez, PR, January 09 - 12, 2007.
Abstract: To be presented at the UPRM Water Microbiology Methods Course, Mayaguez, Puerto Rico, January 9-12, 2007

PUBLISHED REPORT Preliminary Comparative Study of Methods to Extract Virus from Raw and Processed Sewage Sludges 09/28/2007
RHODES, E., B. MCMINN, N. BRINKMAN, J. CASHDOLLAR, AND G. FOUT. Preliminary Comparative Study of Methods to Extract Virus from Raw and Processed Sewage Sludges. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/R-07/118, 2007.
Abstract: Two simple virus extraction techniques were compared to an EPA standard method for detection of human enteric viruses in raw sewage sludge and class A biosolids. The techniques were used to detect both indigenous and seeded virus from a plant that distributes class A material produced by a heat drying process. Virus titers were measured using a plaque assay, a quantal assay and an integrated cell culture-reverse transcription-polymerase chain reaction (ICC-PCR) assay. The best extraction technique overall for detection of indigenous and seeded virus by plaque, quantal, and ICC-PCR assays was a simple chloroform-based technique. The detection of indigenous virus in raw sludge was similar for all three methods by the plaque assay, giving an average of 50±21 PFU/4 grams of sludge. Recovery of seeded virus from raw sludge averaged 0.7% for the EPA versus 22±17% for the other two techniques. Recovery of seeded virus from class A biosolids was similar for all techniques, averaging 95±24%. Both the quantal and the ICC-PCR assays outperformed the plaque assay for virus detection. Virus titers were generally 2->10 fold higher by these assays than by the plaque assay.



 

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