The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2-and 10-liter water samples. When known quantities (10 5 to 10 6 CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 ؋ 10 5 and 2.4 ؋ 10 3 per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 ؋ 10 5 CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10 5 PFU/liter), and phage PP7 (10 5 PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.

Agricultural water is an important source of foodborne pathogens on produce farms. Managing water-associated risks does not lend itself to one-size-fits-all approaches due to the heterogeneous nature of freshwater environments. To improve our ability to develop location-specific risk management practices, a study was conducted in two produce-growing regions to (i) characterize the relationship between Escherichia coli levels and pathogen presence in agricultural water, and (ii) identify environmental factors associated with pathogen detection. Three AZ and six NY waterways were sampled longitudinally using 10-L grab samples (GS) and 24-h Moore swabs (MS). Regression showed that the likelihood of Salmonella detection (Odds Ratio [OR] = 2.18), and eaeA-stx codetection (OR = 6.49) was significantly greater for MS compared to GS, while the likelihood of detecting L. monocytogenes was not. Regression also showed that eaeA-stx codetection in AZ (OR = 50.2) and NY (OR = 18.4), and Salmonella detection in AZ (OR = 4.4) were significantly associated with E. coli levels, while Salmonella detection in NY was not. Random forest analysis indicated that interactions between environmental factors (e.g., rainfall, temperature, turbidity) (i) were associated with likelihood of pathogen detection and (ii) mediated the relationship between E. coli levels and likelihood of pathogen detection. Our findings suggest that (i) environmental heterogeneity, including interactions between factors, affects microbial water quality, and (ii) E. coli levels alone may not be a suitable indicator of food safety risks. Instead, targeted methods that utilize environmental and microbial data (e.g., models that use turbidity and E. coli levels to predict when there is a high or low risk of surface water being contaminated by pathogens) are needed to assess and mitigate the food safety risks associated with preharvest water use. By identifying environmental factors associated with an increased likelihood of detecting pathogens in agricultural

No abstract

No abstract

19 20 21 22 23 produce farms. Managing water-associated risks does not lend itself to one-size-fits-all 24 approaches due to the heterogeneous nature of freshwater environments, and because 25 environmental conditions affect the likelihood of pathogen contamination and the relationship 26 between indicator organism levels (e.g., E. coli) and pathogen presence. To improve our ability 27 to develop location-specific risk management practices, a study was conducted in two produce-28 growing regions to (i) characterize the relationship between E. coli levels and pathogen presence 29 in agricultural water, and (ii) identify environmental factors associated with pathogen detection. 30Three AZ and six NY waterways were sampled longitudinally using 10-L grab samples (GS) and 31 24-h Moore swabs (MS). Regression showed that the likelihood of Salmonella detection (Odds 32 Ratio [OR]=2.18), and eaeA-stx codetection (OR=6.49) was significantly greater for MS 33 compared to GS, while the likelihood of detecting L. monocytogenes was not. Regression also 34 showed that eaeA-stx codetection in AZ (OR=50.2) and NY (OR=18.4), and Salmonella 35 detection in AZ (OR=4.4) were significantly associated with E. coli levels, while Salmonella 36 detection in NY was not. Random forest analysis indicated that interactions between 37 environmental factors (e.g., rainfall, temperature, turbidity) (i) were associated with likelihood of 38 pathogen detection and (ii) mediated the relationship between E. coli levels and likelihood of 39 pathogen detection. Our findings suggest that (i) environmental heterogeneity, including 40 interactions between factors, affects microbial water quality, and (ii) E. coli levels alone may not 41 be a suitable indicator of the food safety risks. Instead, targeted methods that utilize 42 environmental and microbial data (e.g., models that use turbidity and E. coli levels to predict 43 when there is a high or low risk of surface water being contaminated by pathogens) are needed to 44 assess and mitigate the food safety risks associated with preharvest water use. By identifying 45 environmental factors associated with an increased likelihood of detecting pathogens in 46 agricultural water, this study provides information that (i) can be used to assess when pathogen 47 contamination of agricultural water is likely to occur, and (ii) facilitate development of targeted 48 interventions for individual water sources, providing an alternative to existing one-size-fits-all 49 approaches. 50 51 52 3 3Preharvest surface water use for produce production (e.g., irrigation, fertigation, pesticide 53 application, dust abatement) has repeatedly been identified as a factor associated with an 54

Biosolids contain a wide variety of organic contaminants that are known for their ability to inhibit PCR. During sample processing, these contaminants are coconcentrated with microorganisms. Elevated concentrations of these compounds in concentrates render samples unsuitable for molecular applications. Glycine-based elution and recovery methods have been shown to generate samples with fewer PCR inhibitory compounds than the current U.S. EPA-recommended method for pathogen recovery from biosolids. Even with glycine-based methods, PCR inhibitors still persist in concentrations that may interfere with nucleic acid amplification. This results in considerable loss of time and resources and increases the probability of false negatives. A method to estimate the degree of inhibition prior to application of molecular methods is desirable. Here we report fluorescence excitation-emission matrix (EEM) profiling as a tool for predicting levels of molecular inhibition in sample concentrates of biosolids.

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean ؎ standard deviation, 47.53 ؎ 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean ؎ standard deviation, 436 ؎ 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.Cryptosporidium and Giardia are enteric parasites that infect a wide range of vertebrate hosts, including birds and mammals. Cryptosporidium parvum and Giardia lamblia are the most common pathogenic species of these parasites in humans. In humans, these organisms can cause persistent diarrhea for 1 to 3 weeks. One of the most common modes of transmission is consumption of feces-contaminated water (17,25,34). It is estimated that 80 to 96% of surface waters in the United States are contaminated with Cryptosporidium and Giardia (16,29).

Increasing demands on water resources have made water reuse an attractive option for extending water supplies in the southwest. However, concerns remain about the potential risks of contact with recycled water. This study focused on perceptions regarding water reuse and how these may affect future utilization of the resource. This study, based on a telephone survey of 400 randomly-selected Arizona residents, was used to assess public opinion of water reuse in the state. Survey results indicated that residents feel it is important for their community to use recycled water. In fact, 76% of those surveyed support using 'consumer incentives for using recycled water', and over two-thirds of respondents support 'increasing water or sewer rates to treat water to higher standards'. Despite this support, the survey revealed that almost two-thirds of the respondents have concerns about recycled water. Those concerns can be alleviated by providing 'better information about recycled water'. Education level proved to be the most significant demographic affecting perception of terminology and recycled water uses. These results can be used by water agencieseven those outside Arizonato address community concerns, effectively promote water reuse, and develop more sustainable and accepted alternatives to augment their water portfolios.