Analysis of groundwater for the presence of microbiota offers researchers a way to estimate the potential for Giardia and Cryptosporidium contamination.A total of 383 groundwater samples from 166 different sites were analyzed for Cryptosporidium, Giardia, and other microbiota (1–500 μm) using immunofluorescence assay and light microscopy procedures. Analysis of the data showed 11 percent of the sites (19 of 166) were sometimes positive for Cryptosporidium, Giardia, or both. Statistical analyses determined that microbiota in eight major groups were indicative of Cryptosporidium and Giardia contamination. These groups include algae, arthropods, rotifers, fungi, nematodes, colorless flagellates, amoebae, and gastrotrichs. Cryptosporidium and Giardia occurrence also correlated positively with the three risk categories developed by the US Environmental Protection Agency to rank direct surface water influence on groundwaters (p = 0.001). However, the specific numerical scores used to determine risk categories provided no better prediction of the pathogens than the three general categories. To determine vulnerability to contamination, researchers first should investigate those groundwater sources with a diversity of microbiota.

Effective application of Cryptosporidium research is tied to adequate documentation of oocyst quality and enumeration techniques. Noting the dearth of accurate information regarding the quality and quantity of Cryptosporidium oocysts used for experimentation, the authors used isolates meeting stringent quality assurance criteria to document the variation of results with three different enumeration techniques—hemacytometer, cellulose–acetate membrane, and well slide. In 70 comparisons of the three techniques, results generated by well slide and hemacytometer varied by an average of 24.7 percent, hemacytometer and membrane results varied 67.6 percent, and well‐slide and membrane results varied 79.3 percent. Significant discrepancies between counts generated by different techniques indicate a strong probability of poor accuracy in previous enumeration‐based studies. Recovery of oocysts after Percoll‐sucrose flotation varied considerably with either hemacytometer or membrane‐counting techniques, which helps explain low precision with the Information Collection Rule protozoan method. Incomplete description of experimental procedures hinders Cryptosporidium research directed toward improving the analytical method, evaluation of water treatment efficacy, and surrogate development.

Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.Cryptosporidium parvum, an intestinal protozoan parasite, continues to be an important cause of waterborne gastrointestinal disease worldwide. Due to oocyst robustness consisting of environmental stability and resistance to normal water disinfection processes (14,22), the low infectious dose (10), and the lack of chemotherapy (23), detection and enumeration of this organism in both treated and untreated waters have become a focus of the water industry in order to prevent future incidents of cryptosporidiosis.The currently accepted technique for oocyst recovery from water samples is immunomagnetic separation (IMS)-fluorescent antibody (FA) detection, as described by United States Environmental Protection Agency (EPA) Method 1623 (25). The reported IMS-FA recovery rates for oocysts seeded into previously concentrated water pellets of various turbidities have been 62 to 100% (oocyst seed density as determined by dilution, 36 to 976) (20), 55.9 to 83.1% (oocyst seed density as determined by dilution, 89.1 to 98.7) (17), 68 to 83% (oocyst seed density as determined by dilution, 525 to 870) (3), and 84.3% (oocyst seed density as determined by flow cytometry, 100) (19). In an additional study, in which oocysts were seeded into 10-liter grab samples of various turbidities, the reported recoveries ranged from Ͻ1.7 to 56.6% (oocyst seed densities, 1,615 and 2,880) (8).However, the criteria for o...

Knowledge of microbiotic particulate composition provides valuable information for assessing treatment plant performance and enhancing plant operation. Concentration of microbiota and total particulates in raw and finished water samples from 55 conventional treatment plants was compared. Analyses included (1) enumeration and identification of the microbiota, (2) direct measurement of the pellet volume of total particulates after laboratory centrifugation, (3) total particle counts, and (4) Giardia and Cryptosporidium detection. Organism reduction based on microscopic particulate analysis in the raw and finished waters ranged from –0.8 to 6.0 log. There was a significant correlation between reduction values generated by organism counts, centrifugate pellet measurements, and particle counts. However, only organism reduction values remained consistent regardless of influent turbidity or flocculent material present in the effluent. Because of low concentrations and intermittent occurrence, counts of Giardia and Cryptosporidium did not accurately reflect treatment plant performance.

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