Measurement of Cryptosporidium parvum oocysts in: Supernatant, sludge and field inactivation trials.

摘要

There are three aspects of this research: (1) developing a molecular-based method of detection and quantitation of Cryptosporidium parvum oocysts, using quantitative polymerase chain reaction (QPCR), (2) using this QPCR method to estimate the number of oocysts in supernatant and sludge samples after conventional water treatment processes, and (3) examining the fate of oocysts in sludge exposed to agricultural land.;QPCR was developed by using a MIMIC template to generate a standard curve, which was used to determine the final estimate of oocysts in the sludge and supernatant samples. PCR existing methods were modified to increase the sensitivity and specificity of the test.;The conventional water treatment method was used to treat raw water spiked with 1.67 x 105 oocysts/Liter. Oocysts in the sludge and supernatant samples were extracted, purified, and estimated using QPCR. A mass balance was used to determine the number of oocysts lost in the experiment.;Sentinel chambers containing 1.0 g of mixture of soil sludge were spiked with 2.5 x 105 oocysts and buried in 10cm soil along with the controls. Soil temperature was monitored daily. The die-off rates of oocysts were monitored approximately every fifteen days. A mass balance was used to determine the number of oocysts lost in the experiment.;Less than 20 percent of oocysts were recovered from the sludge samples and greater than 83 percent of oocysts were recovered from the supernatant samples in 16 experiments conducted in triplicate. Percent of oocysts lost in the treatment was 0.03 percent. Based on the standard curve, QPCR detection limit was one oocysts/2liter sample.;During the field inactivation trials, 49.3 percent of the oocysts remain viable after 60 days inactivation. The mean inactivation rate of oocysts in the sentinel chambers after 17, 30, 45, and 60 days were 0.0045, 0.0033, 0.0043, and 0.012 day-1. Also, the mean inactivation rates of oocysts in the control units after 0.0023, 0.0019, 0.0017, and 0.0025 day -1. An averaged 4.8 percent of oocysts were lost in the experiments. Overall, the study demonstrated presence of viable oocysts in the sludge samples, which could potentially be used to fertilize agricultural land.