Effects of pH and Magnetic Material on Immunomagnetic Separation of
            <i>Cryptosporidium</i>
            Oocysts from Concentrated Water Samples

213

Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States. The UF-based procedure included hollow-fiber UF as the primary step for concentrating microbes and then used membrane filtration for bacterial culture assays, immunomagnetic separation for parasite recovery and quantification, and centrifugal UF for secondary concentration of viruses. Water samples were tested for nine water quality parameters to investigate whether water quality data correlated with measured recovery efficiencies and molecular detection levels. Average total method recovery efficiencies were 71, 97, 120, 110, and 91% for X174 bacteriophage, MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts, respectively. Realtime PCR and reverse transcription-PCR (RT-PCR) for seeded microbes and controls indicated that tap water quality could affect the analytical performance of molecular amplification assays, although no specific water quality parameter was found to correlate with reduced PCR or RT-PCR performance.

Section: Discussioncontrasting

confidence: 42%

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confidence: 99%

Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric Microbes in 100-Liter Tap Water Samples

Hill

Kahler

Jothikumar

et al. 2007

213

“…Many investigators found that turbidity was related to recoveries of oocysts (4,5,7,15,26). The efficiency of the IMS technique was shown to decrease when the adjusted pH deviated from 7.0 (16) or when dissolved iron concentrations were greater than 4 mg/liter (38). In contrast, in the Information Collection Rule Supplemental Survey (ICRSS), wherein 430 samples were collected from 87 source waters, all measured water quality properties, including turbidity, were found to be unrelated or weakly related to recoveries of oocysts (32).…”

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confidence: 99%

Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water by Using U.S. Environmental Protection Agency Method 1623

Francy

Simmons

Ware

et al. 2004

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r ‫؍‬ 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.

“…Although cryptosporidiosis is mainly confined to young individuals, low-level asymptomatic infections in postweaned and adult cattle have been reported (6), with up to 10 4 oocysts per g of feces excreted (17). In addition, postparturient ewes may shed increased but low concentrations of C. parvum oocysts (100 to 5,700 oocysts g Ϫ1 ) (21).…”

mentioning

confidence: 99%

“…However, an attempt to improve recovery by using EDTA to sequester metal ions that may compete for bead binding sites was unsuccessful. Recent studies have demonstrated that the pH during oocyst capture is an important factor that affects the recovery of oocysts by IMS (10) and that the levels of recovery of oocysts from deionized water at pH 7.0 were significantly higher than those from water that deviated as little as 0.12 pH unit from the optimum. In the present study, deviation of the pH from the optimum during the IMS procedure was not considered to be the cause of poor recovery of oocysts from pig feces, since when it was checked, the pH before and after IMS deviated little from the optimum (data not shown).…”

mentioning

confidence: 99%

Recovery and Enumeration of Cryptosporidium parvum from Animal Fecal Matrices

Davies

Kaucner

Deere

et al. 2003

Accurate quantification of Cryptosporidium parvum oocysts in animal fecal deposits on land is an essential starting point for estimating watershed C. parvum loads. Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation. The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types. The levels of recovery of 10 2 oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35%. The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50%. The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data. Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos. Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery. Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces ؊1 must be present to be detected.The protozoan parasite Cryptosporidium parvum is a major cause of waterborne diarrheal illness in human and animal populations. The transmissive form of this organism, the oocyst, frequently finds its way into surface waters, either directly through sewage discharge or indirectly in runoff from land grazed by livestock or wildlife. Oocysts present in animal fecal deposits on land are clearly responsible for many event-related increases in the concentrations of in-stream pathogens in watersheds (3, 8). However, there are significant knowledge gaps regarding the mechanisms and magnitude of pathogen transport and the effects of environmental factors on oocyst inactivation (7). In particular, there have been few attempts to predict the fate of oocysts deposited on land by using quantifiable parameters. The responsible authorities are advocating adequate watershed management as a fundamental requirement for reducing pathogen risks to drinking water quality, and the United States Environmental Protection Agency is seeking estimates of total maximum daily loads for watershed pathogens (18). The quantification of transport mechanisms and environmental inactivation for key pathogens should enable models to be constructed that can be used to predict source water quality in order to better manage the factors that govern p...