A survey of enteric viruses and indicator bacteria was carried out in eight community water supply sources (four wells and four springs) in East Tennessee. Seven sites derived their water from carbonate aquifers and one from fractured sandstone. Four of the sites were deemed "low-risk" based on prior monitoring of fecal indicators and factors such as presence of thick layers of overlying sediments. The remaining sites were deemed "high-risk." Enteric viruses (enterovirus and reovirus) were detected by cell culture at least once in seven of the eight wells or springs including all but one of the four low-risk sites. Viral RNA, however, was not detected in any of the samples by reverse transcription-polymerase chain reaction. Conventional indicators of microbial contamination (Escherichia coli and total coliform bacteria) were detected together with culturable viruses in seven of nine virus positive samples. Bacteroides, an alternative fecal indicator which has not previously been used in groundwater investigations, was also detected in all but one of the samples containing E. coli or total coliform bacteria, as well as in one sample where viruses were present in the absence of other bacterial indicators. The study highlights some of the challenges involved in surveys of virus occurrence and indicates that culturable enteric viruses in East Tennessee karst aquifers may be more widespread than previously observed in studies of karst aquifers in Pennsylvania (8%), the Ozark region of Missouri (< 1%), or several other states covered in a national microbial water quality survey conducted by the U.S. Environmental Protection Agency (43%).

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Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12 . The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.

Aim USEPA Method 1623, or its equivalent, is currently used to monitor for protozoan contamination of surface drinking water sources worldwide. At least three approved staining kits used for detecting Cryptosporidium and Giardia are commercially available. This study focuses on understanding the differences among staining kits used for Method 1623. Methods and Results Merifluor and EasyStain labeling kits were used to monitor Cryptosporidium oocyst and Giardia cyst densities in New York City’s raw surface water sources. In the year following a change to the approved staining kits for use with Method 1623, an anomaly was noted in the occurrence of Giardia cysts in New York City’s raw surface water. Specifically, Merifluor-stained samples had higher Giardia cyst densities as compared with those stained with EasyStain. Side by side comparison revealed significantly lower fluorescence intensities of Giardia muris as compared with G. duodenalis cysts when labeled with EasyStain. Conclusions This study showed very poor fluorescence intensity signals by EasyStain on G. muris cysts results in lower cyst counts, while Merifluor, with its broader Giardia cyst staining specificity, results in higher cyst counts, when using Methods 1623. Significance and Impact of Study These results suggest that detected Giardia cyst concentrations are dependent on the staining kits used, which can result in a more or less conservative estimation of occurrences and densities of zoonotic Giardia cysts by detecting a broader range of Giardia species/Assemblages.

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