Effects of time and watershed characteristics on the concentration of Cryptosporidium oocysts in river water.

Water samples were collected from four locations on two rivers in Washington State and analyzed by membrane filtration-immunofluorescence assay to establish Cryptosporidium oocyst concentrations. Sampling locations were selected to evaluate effects of watershed character, from pristine mountain to downstream agricultural, on oocyst concentrations. Samples were collected at six biweekly intervals from late June to early September, with two additional sets of five samples taken on separate days (one set taken at bihourly intervals and one set taken simultaneously). Cryptosporidium oocysts were found in 34 of 35 samples at concentrations ranging from about 0.2 to 65 oocysts per liter. Oocyst concentrations were highest early in the sampling period, when they were influenced by postrainfall runoff, and decreased through the summer months. Oocyst concentrations found in ten samples collected on two days (5 samples per day) showed no short-term variations. Oocyst concentrations and oocyst production per square mile (ca. 2.6 km2) of watershed found in water draining a controlled public water supply watershed were the lowest observed. The concentrations and production rates for drainage from an adjacent, comparable, but uncontrolled watershed were nearly 10 times higher. The concentration and production rates of the downstream area influenced by dairy farming were nearly 10 times higher than rates at the upstream stations. The data showed clearly that oocyst concentrations were consistently observed above the detection limit of the analytical method, about 0.1 oocysts per liter; that oocyst concentrations were continuous as opposed to intermittent; and that watershed character and management affected surface water oocyst concentrations significantly.     

Identification of Cryptosporidium oocysts in river water.

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.     

Identification of Cryptosporidium oocysts in river water

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.     

Contamination of river water by Cryptosporidium parvum oocysts in western Japan

In Japan, only a few rivers have been inspected for Cryptosporidium parvum contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for Cryptosporidium oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for Cryptosporidium oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the C. parvum found as the bovine or human type. C. parvum was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained C. parvum oocysts. The number of C. parvum oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the C. parvum detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with C. parvum oocysts, as reported in other countries.     

A new method for the concentration of Cryptosporidium oocysts from water

A novel method for the concentration of Cryptosporidium oocysts from water has been developed, based upon the precipitation of calcium carbonate. A 10 1 water sample is treated by adding solutions of calcium chloride and sodium bicarbonate and raising the pH value to 10 with sodium hydroxide. Crystals of calcium carbonate form and enmesh particles in the Cryptosporidium oocyst size range. The crystals are allowed to settle, the supernatant fluid is discarded and the calcium carbonate precipitate dissolved in sulphamic acid. The sample can be concentrated further by centrifugation. Recoveries of oocysts from seeded samples of deionized, tap and river water were in excess of 68%.     

Effects of combined water potential and temperature stresses on Cryptosporidium parvum oocysts.

Hosts infected with the parasite Cryptosporidium parvum may excrete oocysts on soils in watersheds that supply public drinking water. Environmental stresses decrease the numbers of oocysts after deposition on soils. However, the rates and effects of combined stresses have not been well characterized, especially for the purposes of estimating decrease in numbers. We subjected oocysts to combined stresses of water potential (-4, -12, and -33 bars), above-freezing temperatures (4 and 30 degrees C), and a subfreezing temperature (-14 degrees C) for 1, 14, and 29 days and one to six freeze-thaw cycles (-14 to 10 degrees C) to estimate coefficients to characterize population degradation using multiplicative error and exponential decay models. The experiments were carried out in NaCl solutions with water potentials of -4, -12, and -33 bars, in combination with temperature stresses at levels that could be expected in natural soils. Increased water potential increased the rate of population degradation for all temperature conditions investigated. Enhanced degradation leads to estimated rates of population degradation that are greater than those that have been reported and used in previous studies conducted to assess risk of water supply contamination from sources of C. parvum.     

Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern Spain

AIMS: To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. METHODS AND RESULTS: Samples from five sewage treatment plants were taken monthly and quarterly during 2003. In addition, water was collected monthly from the River Llobregat (NE Spain) during the period from 2001 to 2003. All samples were analysed by filtration on cellulose acetate filters or through Envirocheck using EPA method 1623, followed by immunomagnetic separation and examination by laser scanning cytometry. All raw sewage, secondary effluent and river water samples tested were positive for Cryptosporidium oocysts. Of the tertiary sewage effluents tested, 71% were positive for Cryptosporidium oocysts. The proportion of viable oocysts varied according to the sample. CONCLUSIONS: Two clear maxima were observed during spring and autumn in raw sewage, showing a seasonal distribution and a correlation with the number of cryptosporidiosis cases and rainfall events. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first data on the occurrence of Cryptosporidium oocysts in natural waters in north-eastern Spain.     

Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine.

Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2 microm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3); pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log(10).day(-1)) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters.     

Effect of sample holding time on recovery of Cryptosporidium oocysts and Giardia cysts from water samples

U.S. Environmental Protection Agency methods for analysis of water for Cryptosporidium and Giardia stipulate maximum sample holding times which are not always practical to comply with. A spiking experiment indicated that holding times of up to 2 weeks had no significant effect on recovery of these parasites from 10-liter samples of raw water in plastic carboys.     

Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.     

Effect of pasteurization on infectivity of Cryptosporidium parvum oocysts in water and milk.

Cryptosporidium parvum is a major cause of diarrheal disease in humans and has been identified in 78 other species of mammals. The oocyst stage, excreted in feces of infected humans and animals, has been responsible for recent waterborne outbreaks of human cryptosporidiosis. High temperature and long exposure time have been shown to render oocysts (suspended in water) noninfectious, but for practical purposes, it is important to know if high-temperature--short-time conditions (71.7 degrees C for 15 s) used in commercial pasteurization are sufficient to destroy infectivity of oocysts. In this study, oocysts were suspended in either water or whole milk and heated to 71.7 degrees C for 15, 10, or 5 s in a laboratory-scale pasteurizer. Pasteurized and nonpasteurized (control) oocysts were then tested for the ability to infect infant mice. No mice (0 of 177) given 10(5) oocysts pasteurized for 15, 10, or 5 s in either water or milk were found to be infected with C. parvum on the basis of histologic examination of the terminal ileum. In contrast, all (80 of 80) control mice given nonpasteurized oocysts were heavily infected. These data indicate that high-temperature--short-time pasteurization is sufficient to destroy the infectivity of C. parvum oocysts in water and milk.     

Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water.

Cryptosporidium parvum oocysts suspended in 0.5 ml of distilled water were pipetted into plastic vials which were inserted into wells in the heated metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 60 to 100 degrees C. At each temperature setting four vials containing C. parvum oocysts were placed into wells and held for 15 s before time was recorded as zero, and then pairs of vials were removed 1 and 5 min later. Upon removal, all vials were immediately cooled on crushed ice. Also, at each temperature interval one vial containing 0.5 ml of distilled water was placed in a well and a digital thermometer was used to record the actual water temperature at 30-s intervals. Heated oocyst suspensions as well as unheated control suspensions were orally inoculated by gavage into 7- to 10-day-old BALB/c mouse pups to test for infectivity. At 96 h after inoculation the ileum, cecum, and colon from each mouse were removed and prepared for histology. Tissue sections were examined microscopically. Developmental-stage C. parvum was found in all three gut segments from all mice that received oocysts in unheated water and in water that reached temperatures of 54.4, 59.9, and 67.5 degrees C at 1 min when vials were removed from the heat source. C. parvum was also found in the ileum of one of six mice that received oocysts in water that reached a temperature of 59.7 degrees C at 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of surface characteristics on the stability of Cryptosporidium parvum oocysts

Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems.     

Implications of biofilm-associated waterborne Cryptosporidium oocysts for the water industry

Waterborne Cryptosporidium has been responsible for drinking water-associated disease outbreaks in a number of developed countries. As a result of the resistance of Cryptosporidium to chlorine, which is typically applied as a final barrier to protect the quality of distributed drinking water, current management practices are focused on source-water management and water treatment as ways of preventing Cryptosporidium from entering drinking-water supplies. In the event that treatment barriers fail, surprisingly little is known of the fate of oocysts once they enter a distribution system. To assess properly the risks of waterborne Cryptosporidium, a more thorough understanding of the fate of oocysts in water distribution systems, with emphasis on Cryptosporidium-biofilm interactions, is required.     

Routine monitoring of Cryptosporidium oocysts in water using flow cytometry

A flow cytometric method for the routine analysis of environmental water samples for the presence of Cryptosporidium oocysts has been developed. It uses a Coulter Epics Elite flow cytometer to examine water samples and to separate oocysts from contaminating debris by cell sorting. The sorted particles are then rapidly screened by microscopy. The method has been evaluated and compared with direct epifluorescence microscopy on 325 river, reservoir and drinking water samples. The technique was found to be more sensitive, faster and easier to perform than conventional epifluorescent microscopy for the routine examination of water samples for Cryptosporidium.     

Effects of pH and magnetic material on immunomagnetic separation of Cryptosporidium oocysts from concentrated water samples

In this study, we examined the effect that magnetic materials and pH have on the recoveries of Cryptosporidium oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted.     

Molecular fingerprinting of Cryptosporidium oocysts isolated during water monitoring

We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.     

Inactivation of Cryptosporidium parvum oocysts in water using ultrasonic treatment

Ultrasound in a liquid phase cause mass and heat transfer across the liquid through cavitational processes which act as nanoreactors to generate unstable mechanical equilibrium. The effect of 1 MHz ultrasound on the inactivation of Cryptosporidium parvum was investigated. Continuous irradiation of ultrasound (20 min) increased temperature due to cavitational phenomena. Ultrasound irradiation of liquid containing C. parvum showed significant quantitative changes in pH, temperature and inactivation of C. parvum (102.7 oocysts killed/s) with a minimum energy consumption (0.05 oocysts/s).     

Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water.

The accurate determination of the presence of Giardia cysts and Cryptosporidium oocysts in surface waters requires a reliable method for the detection and enumeration of these pathogenic organisms. Published methods have usually reported recovery efficiencies of less than 50% for both cysts and oocysts. Typically, the losses are greater for Cryptosporidium oocysts than they are for Giardia cysts. The purpose of this study was to examine procedures used for sample collection, elution, concentration, and clarification to determine when losses of cysts and oocysts occurred during processing. The results showed that major losses of cysts and oocysts occurred during centrifugation and clarification. Depending on the centrifugation force, oocyst losses of as high as 30% occurred for each centrifugation step. A 1.15-specific-gravity Percoll-sucrose gradient was needed to optimize recovery of oocysts from natural water samples. Minor improvements in the procedure could be accomplished by selecting a filter other than the recommended 1-micron-pore-size (nominal-porosity) polypropylene filter.     

Gaseous disinfection of Cryptosporidium parvum oocysts.

Purified oocysts of Cryptosporidium parvum suspended in approximately 400 microliters of phosphate-buffered saline or deionized water in microcentrifuge tubes were exposed at 21 to 23 degrees C for 24 h to a saturated atmosphere of ammonia, carbon monoxide, ethylene oxide, formaldehyde, or methyl bromide gas. Controls were exposed to air. Oocysts in each tube were then rinsed and resuspended in fresh, deionized water, and 1 million oocysts exposed to each gas were orally administered to each of three to six neonatal BALB/c mice in replicate groups. Histologic sections of ileum, cecum, and colon tissues taken from each mouse 72 h after oral administration of oocysts were examined microscopically to determine if infection had been established. All 15 mice given oocysts exposed to carbon monoxide had numerous developmental stages of cryptosporidium in all three intestinal segments. Of 10 mice given oocysts exposed to formaldehyde, 6 had a few developmental stages of cryptosporidium in the ileum. No mice given oocysts exposed to ammonia, ethylene oxide, or methyl bromide were found to be infected. These findings indicate the efficacy of these low-molecular-weight gases (ammonia, ethylene oxide, and methyl bromide) as potential disinfectants for C. parvum oocysts where soil, rooms, buildings, tools, or instruments might be contaminated.