Recovery and Enumeration of Cryptosporidium parvum from Animal Fecal Matrices

Abstract

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...

Prevalence of Cryptosporidium and other intestinal parasites using different diagnostic techniques in Enugu Metropolis, South-Eastern Nigeria

“…The findings in this study showed higher detection of infectious agents in stool including Cryptosporidium in 6 samples, yeasts in 24 samples and B. hominis in 1 sample (Figure 2). These findings are consistent with the scientific reports of Davies et al (2003) and Weber et al (1992) on this technique as indicated by the extra detection of 7% and 10% of Cryptosporidium and yeasts, respectively (Figure 3).…”

“…Studies have shown that this method is efficient in the detection of parasites even when they appear in low numbers in a specimen (Weber et al, 1992;Davies et al, 2003;Cheesbrough, 2005). The findings in this study showed higher detection of infectious agents in stool including Cryptosporidium in 6 samples, yeasts in 24 samples and B. hominis in 1 sample (Figure 2).…”

Concentrations of Pathogens and Indicators in Animal Feces in the Sydney Watershed

“…A 0.5-g aliquot of the fecal sample was weighed into a sterile 50-ml centrifuge tube. Diethyl ether was used in dispersal and defatting of calf fecal samples as described by Davies et al (9). To quantify recovery efficiency, each individual sample was seeded with ColorSeed (Biotechnology Frontiers Ltd.…”

Dispersion and Transport ofCryptosporidiumOocysts from Fecal Pats under Simulated Rainfall Events

“…Ltd., Castle Hill, NSW, Australia). The membranes were mounted on glass slides and scanned at ϫ250 magnification as described by Davies et al (14). Runoff samples from the pat experiments generally did not require concentration by ultrafiltration, but portions (10 ml) of these samples were assayed directly by IMS.…”

“…However, there are difficulties associated with estimating the prevalence of pathogen infections in animals. These problems include whether to sample animals or their scats (25), how to calculate pathogen concentrations and prevalence from fecal pat data (12), and the detection limits of the methods used for enumeration (14,40).…”