The drinking water and wastewater industries are interested in developing a better understanding of sources of fecal contamination. Microbial source tracking (MST) offers the potential to apportion the contribution of various animal groups to this contamination. However, while there are many methods available there is no clear indication as to the most appropriate approach. There has been little systematic comparison of methods and only a few blind trials, and issues of reproducibility, accuracy, sensitivity, specificity, resolution, and robustness need to be addressed. There is confusion among potential end-users and some disagreement among scientists. Therefore, due to the uncertainty surrounding the most appropriate tools and applications of MST, a workshop of 45 experts representing water and wastewater utilities, academia, state and federal government agencies, medical institutions, and private laboratories was convened in San Antonio, Texas over a three-day period (February 16 – 18, 2005). The overall objective of the workshop was to identify the knowledge gaps and research needs for application of MST technologies by the wastewater and drinking water industries.

This study investigates the potential for biological repair in UV-irradiated oocysts, and it presents the results of concurrent lines of investigation to examine whether reactivation occurs following UV disinfection and also to determine if C. parvum has the genetic basis for repair. The primary objectives of the study included the following: Determine whether C. parvum oocysts of multiple isolates, irradiated with varying doses of UV light, can repair and regain infectivity under light or dark conditions using in-vitro cell culture with a human cell line.   Confirm cell culture results with animal infectivity using a neonatal mouse model.   Identify possible DNA repair genes in C. parvum by performing homology searches with sequence databases, polymerase chain reaction (PCR) assays, and sequencing clones from a genomic library.   Measure the extent of UV-induced DNA damage by quantitative PCR.   If reactivation is detected, evaluate the effects of both post-UV chemical disinfection on oocyst reactivation and determine the increased UV dosage necessary to inhibit repair. Originally published by AwwaRF for its subscribers in 2004.

Cell culture techniques are routinely used for measuring the infectivity of a wide range of human pathogens. A variety of different cell culture systems and detection methodologies have been applied to Cryptosporidium parvum. However, the correlation between cell culture methods and animal infectivity assays has not been thoroughly investigated. Although many cell culture methods have been developed for C. parvum, it has not been proven that infectivity in cell culture is a good indicator of the ability of oocysts to cause infections in animals. The objective of this research was to compare in-vitro cell culture methods with a mouse assay for measuring infectivity of C. parvum?oocysts. The specific objectives were to (1) compare the dose response and sensitivity of cell culture and mouse assays with multiple isolates; (2) compare infectivity methods with oocysts exposed to environmental water samples; (3) determine the reproducibility and variability of the methods; and (4) compare cell culture and animal assays for assessing ozone and UV disinfection.For untreated oocysts, challenge doses were enumerated by flow cytometry. Dose response curves were constructed by regression analysis of oocyst dose against a logistic transformation of the proportional infectivity and the 50% infectious doses for each isolate were calculated by solving the regression for a logit value of zero. Infections in CD-1 mice were detected by microscopy following staining with hematoxylin and eosin. Infection in HCT-8 and Caco-2 cells was detected by C. parvum-specific RT-PCR. In MDCK cells, infection was detected using immunofluorescence. For disinfection studies, oocysts were exposed to UV using a medium-pressure, collimated beam apparatus and inactivation was measured as the difference in ID50 of unexposed and UV-exposed oocysts. Oocysts were exposed to ozone using batch, semi-batch, and single continuously stirred tank reactors at 1, 5, and 15?C.This investigation demonstrated that in-vitro cell culture was equivalent with a mouse assay for measuring infectivity of untreated C. parvum?oocysts and should therefore be considered a practical alternative for assessing the potential of oocysts to cause infection. However, the high levels of variability displayed by mouse and cell culture methods indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences. Of the three cell culture assays, the HCT-8/RT-PCR method displayed the closest agreement with the CD-1 mouse assay. C. hominis?was infectious in HCT-8 cells but did not infect mice. Similar results were obtained with CD-1 mice and HCT-8 cells for measuring infectivity of oocysts that had been exposed to environmental water for 35 days. There was also very good agreement between HCT-8 cell culture and CD-1 mouse assays for measuring UV inactivation of C. parvum. A medium-pressure UV dosage of 5.6 mJ/cm2 resulted in 2-log10 inactivation. The shapes of ozone inactivation curves were generally the same for mouse and cell culture derived data although the CD-1 mouse assay typically generated 0.5 to 1-log10 higher levels of inactivation than HCT-8 cells. In addition, there was a stimulatory response in oocysts exposed to ozone below 20 mg.min/L when assayed by HCT-8 cell culture. Consequently, further research is necessary to understand the response of oocysts to ozone when inactivation is assessed by cell culture methods. The water industry should adopt in-vitro cell culture as a routine method for measuring the infectivity of waterborne C. parvum?and C. hominis?oocysts. This project has demonstrated that cell culture has equivalency with the standard CD-1 mouse assay and cell culture assays can be applied to oocysts recovered from water using approved methods. However, there needs to be a thorough, robust, and well-controlled study to compare the various cell culture-based assays for measuring C. parvum?and C. hominis?infectivity. This evaluation should include inter-laboratory comparisons and round-robin testing. Cell culture-based assays should also be used to assess disinfection of C. hominis?isolates. Originally published by AwwaRF for its subscribers in 2004.?This publication can also be purchased and downloaded via Pay Per View on Water Intelligence Online - click on the Pay Per View icon below

The drinking water and wastewater industries are interested in developing a better understanding of sources of fecal contamination. Microbial source tracking (MST) offers the potential to apportion the contribution of various animal groups to this contamination. However, while there are many methods available there is no clear indication as to the most appropriate approach. There has been little systematic comparison of methods and only a few blind trials, and issues of reproducibility, accuracy, sensitivity, specificity, resolution, and robustness need to be addressed. There is confusion among potential end-users and some disagreement among scientists. Therefore, due to the uncertainty surrounding the most appropriate tools and applications of MST, a workshop of 45 experts representing water and wastewater utilities, academia, state and federal government agencies, medical institutions, and private laboratories was convened in San Antonio, Texas over a three-day period (February 16 – 18, 2005). The overall objective of the workshop was to identify the knowledge gaps and research needs for application of MST technologies by the wastewater and drinking water industries.

This study investigates the potential for biological repair in UV-irradiated oocysts, and it presents the results of concurrent lines of investigation to examine whether reactivation occurs following UV disinfection and also to determine if C. parvum has the genetic basis for repair. The primary objectives of the study included the following: Determine whether C. parvum oocysts of multiple isolates, irradiated with varying doses of UV light, can repair and regain infectivity under light or dark conditions using in-vitro cell culture with a human cell line.   Confirm cell culture results with animal infectivity using a neonatal mouse model.   Identify possible DNA repair genes in C. parvum by performing homology searches with sequence databases, polymerase chain reaction (PCR) assays, and sequencing clones from a genomic library.   Measure the extent of UV-induced DNA damage by quantitative PCR.   If reactivation is detected, evaluate the effects of both post-UV chemical disinfection on oocyst reactivation and determine the increased UV dosage necessary to inhibit repair. Originally published by AwwaRF for its subscribers in 2004.

Cell culture techniques are routinely used for measuring the infectivity of a wide range of human pathogens. A variety of different cell culture systems and detection methodologies have been applied to Cryptosporidium parvum. However, the correlation between cell culture methods and animal infectivity assays has not been thoroughly investigated. Although many cell culture methods have been developed for C. parvum, it has not been proven that infectivity in cell culture is a good indicator of the ability of oocysts to cause infections in animals. The objective of this research was to compare in-vitro cell culture methods with a mouse assay for measuring infectivity of C. parvum?oocysts. The specific objectives were to (1) compare the dose response and sensitivity of cell culture and mouse assays with multiple isolates; (2) compare infectivity methods with oocysts exposed to environmental water samples; (3) determine the reproducibility and variability of the methods; and (4) compare cell culture and animal assays for assessing ozone and UV disinfection.For untreated oocysts, challenge doses were enumerated by flow cytometry. Dose response curves were constructed by regression analysis of oocyst dose against a logistic transformation of the proportional infectivity and the 50% infectious doses for each isolate were calculated by solving the regression for a logit value of zero. Infections in CD-1 mice were detected by microscopy following staining with hematoxylin and eosin. Infection in HCT-8 and Caco-2 cells was detected by C. parvum-specific RT-PCR. In MDCK cells, infection was detected using immunofluorescence. For disinfection studies, oocysts were exposed to UV using a medium-pressure, collimated beam apparatus and inactivation was measured as the difference in ID50 of unexposed and UV-exposed oocysts. Oocysts were exposed to ozone using batch, semi-batch, and single continuously stirred tank reactors at 1, 5, and 15?C.This investigation demonstrated that in-vitro cell culture was equivalent with a mouse assay for measuring infectivity of untreated C. parvum?oocysts and should therefore be considered a practical alternative for assessing the potential of oocysts to cause infection. However, the high levels of variability displayed by mouse and cell culture methods indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences. Of the three cell culture assays, the HCT-8/RT-PCR method displayed the closest agreement with the CD-1 mouse assay. C. hominis?was infectious in HCT-8 cells but did not infect mice. Similar results were obtained with CD-1 mice and HCT-8 cells for measuring infectivity of oocysts that had been exposed to environmental water for 35 days. There was also very good agreement between HCT-8 cell culture and CD-1 mouse assays for measuring UV inactivation of C. parvum. A medium-pressure UV dosage of 5.6 mJ/cm2 resulted in 2-log10 inactivation. The shapes of ozone inactivation curves were generally the same for mouse and cell culture derived data although the CD-1 mouse assay typically generated 0.5 to 1-log10 higher levels of inactivation than HCT-8 cells. In addition, there was a stimulatory response in oocysts exposed to ozone below 20 mg.min/L when assayed by HCT-8 cell culture. Consequently, further research is necessary to understand the response of oocysts to ozone when inactivation is assessed by cell culture methods. The water industry should adopt in-vitro cell culture as a routine method for measuring the infectivity of waterborne C. parvum?and C. hominis?oocysts. This project has demonstrated that cell culture has equivalency with the standard CD-1 mouse assay and cell culture assays can be applied to oocysts recovered from water using approved methods. However, there needs to be a thorough, robust, and well-controlled study to compare the various cell culture-based assays for measuring C. parvum?and C. hominis?infectivity. This evaluation should include inter-laboratory comparisons and round-robin testing. Cell culture-based assays should also be used to assess disinfection of C. hominis?isolates. Originally published by AwwaRF for its subscribers in 2004.?This publication can also be purchased and downloaded via Pay Per View on Water Intelligence Online - click on the Pay Per View icon below