Dispersion and transport of Cryptosporidium Oocysts from fecal pats under simulated rainfall events

The dispersion and initial transport of Cryptosporidium oocysts from fecal pats were investigated during artificial rainfall events on intact soil blocks (1,500 by 900 by 300 mm). Rainfall events of 55 mm h(-1) for 30 min and 25 mm h(-1) for 180 min were applied to soil plots with artificial fecal pats seeded with approximately 10(7) oocysts. The soil plots were divided in two, with one side devoid of vegetation and the other left with natural vegetation cover. Each combination of event intensity and duration, vegetation status, and degree of slope (5 degrees and 10 degrees ) was evaluated twice. Generally, a fivefold increase (P < 0.05) in runoff volume was generated on bare soil compared to vegetated soil, and significantly more infiltration, although highly variable, occurred through the vegetated soil blocks (P < 0.05). Runoff volume, event conditions (intensity and duration), vegetation status, degree of slope, and their interactions significantly affected the load of oocysts in the runoff. Surface runoff transported from 10(0.2) oocysts from vegetated loam soil (25-mm h(-1), 180-min event on 10 degrees slope) to up to 10(4.5) oocysts from unvegetated soil (55-mm h(-1), 30-min event on 10 degrees slope) over a 1-m distance. Surface soil samples downhill of the fecal pat contained significantly higher concentrations of oocysts on devegetated blocks than on vegetated blocks. Based on these results, there is a need to account for surface soil vegetation coverage as well as slope and rainfall runoff in future assessments of Cryptosporidium transport and when managing pathogen loads from stock grazing near streams within drinking water watersheds.     

Soil Organic Carbon Loss and Selective Transportation under Field Simulated Rainfall Events

The study on the lateral movement of soil organic carbon (SOC) during soil erosion can improve the understanding of global carbon budget. Simulated rainfall experiments on small field plots were conducted to investigate the SOC lateral movement under different rainfall intensities and tillage practices. Two rainfall intensities (High intensity (HI) and Low intensity (LI)) and two tillage practices (No tillage (NT) and Conventional tillage (CT)) were maintained on three plots (2 m width × 5 m length): HI-NT, LI-NT and LI-CT. The rainfall lasted 60 minutes after the runoff generated, the sediment yield and runoff volume were measured and sampled at 6-min intervals. SOC concentration of sediment and runoff as well as the sediment particle size distribution were measured. The results showed that most of the eroded organic carbon (OC) was lost in form of sediment-bound organic carbon in all events. The amount of lost SOC in LI-NT event was 12.76 times greater than that in LI-CT event, whereas this measure in HI-NT event was 3.25 times greater than that in LI-NT event. These results suggest that conventional tillage as well as lower rainfall intensity can reduce the amount of lost SOC during short-term soil erosion. Meanwhile, the eroded sediment in all events was enriched in OC, and higher enrichment ratio of OC (ERoc) in sediment was observed in LI events than that in HI event, whereas similar ERoc curves were found in LI-CT and LI-NT events. Furthermore, significant correlations between ERoc and different size sediment particles were only observed in HI-NT event. This indicates that the enrichment of OC is dependent on the erosion process, and the specific enrichment mechanisms with respect to different erosion processes should be studied in future.     

Transport of Cryptosporidium parvum Oocysts through Vegetated Buffer Strips and Estimated Filtration Efficiency

Vegetated buffer strips were evaluated for their ability to remove waterborne Cryptosporidium parvum from surface and shallow subsurface flow during simulated rainfall rates of 15 or 40 mm/h for 4 h. Log₁₀ reductions for spiked C. parvum oocysts ranged from 1.0 to 3.1 per m of vegetated buffer, with buffers set at 5 to 20% slope, 85 to 99% fescue cover, soil textures of either silty clay (19:47:34 sand-silt-clay), loam (45:37:18), or sandy loam (70:25:5), and bulk densities of between 0.6 to 1.7 g/cm³. Vegetated buffers constructed with sandy loam or higher soil bulk densities were less effective at removing waterborne C. parvum (1- to 2-log₁₀ reduction/m) compared to buffers constructed with silty clay or loam or at lower bulk densities (2- to 3-log₁₀ reduction/m). The effect of slope on filtration efficiency was conditional on soil texture and soil bulk density. Based on these results, a vegetated buffer strip comprised of similar soils at a slope of ≤20% and a length of ≥3 m should function to remove ≥99.9% of C. parvum oocysts from agricultural runoff generated during events involving mild to moderate precipitation.     

Deposition of Cryptosporidium Oocysts in Streambeds

The transfer of Cryptosporidium oocysts from the surface water to the sediment beds of streams and rivers influences their migration in surface waters. We used controlled laboratory flume experiments to investigate the deposition of suspended Cryptosporidium parvum oocysts in streambeds. The experimental results demonstrate that hydrodynamic interactions between an overlying flow and a sediment bed cause oocysts to accumulate in the sediments and reduce their concentrations in the surface water. The association of C. parvum with other suspended sediments increased both the oocysts' effective settling velocity and the rate at which oocysts were transferred to the sediment bed. A model for the stream-subsurface exchange of colloidal particles, including physical transport and physicochemical interactions with sediment grains, accurately represented the deposition of both free C. parvum oocysts and oocysts that were attached to suspended sediments. We believe that these pathogen-sediment interactions play an important role in regulating the concentrations of Cryptosporidium in streams and rivers and should be taken into consideration when predicting the fate of pathogens in the environment.     

Transport of Cryptosporidium parvum Oocysts in Soil Columns following Applications of Raw and Separated Liquid Slurries

The potential for the transport of viable Cryptosporidium parvum oocysts through soil to land drains and groundwater was studied using simulated rainfall and intact soil columns which were applied raw slurry or separated liquid slurry. Following irrigation and weekly samplings over a 4-week period, C. parvum oocysts were detected from all soil columns regardless of slurry type and application method, although recovery rates were low (<1%). Soil columns with injected liquid slurry leached 73 and 90% more oocysts compared to columns with injected and surface-applied raw slurries, respectively. Among leachate samples containing oocysts, 44/72 samples yielded viable oocysts as determined by a dye permeability assay (DAPI [4',6'-diamidino-2-phenylindole]/propidium iodide) with the majority (41%) of viable oocysts found in leachate from soil columns with added liquid slurry. The number of viable oocysts was positively correlated (r = 0.63) with the total number of oocysts found. Destructively sampling of the soil columns showed that type of slurry and irrigation played a role in the vertical distribution of oocysts, with more oocysts recovered from soil columns added liquid slurry irrespective of the irrigation status. Further studies are needed to determine the effectiveness of different slurry separation technologies to remove oocysts and other pathogens, as well as whether the application of separated liquid slurry to agricultural land may represent higher risks for groundwater contamination compared to application of raw slurry.     

Inactivation of Cryptosporidium parvum Oocysts by Ammonia

The survival of Cryptosporidium parvum oocysts in soil and water microhabitats may be affected by the environmental production and release of free ammonia. The objective of this study was to determine the effects of increasing free ammonia concentrations and times of exposure on oocyst viability. Wild-type oocysts were obtained from naturally infected calf feces by chemical (continuous-flow) centrifugation and sucrose gradients. Ammonia (NH₃) from a commercial solution was applied in concentrations ranging from 0.007 to 0.148 M. Exposure times ranged from 10 min to 24 h at a constant temperature of 24 ± 1°C. Viability of oocysts was determined with a dye permeability assay and an in vitro excystation assay (M. B. Jenkins, L. J. Anguish, D. D. Bowman, M. J. Walker, and W. C. Ghiorse, Appl. Environ. Microbiol. 63:3844–3850, 1997). Even the lowest concentration of ammonia decreased significantly the viability of oocysts after 24 h of exposure. Increasing concentrations of ammonia increased inactivation rates, which ranged from 0.014 to 0.066 h⁻¹. At the highest concentration of ammonia, a small fraction of viable oocysts still remained. Exposure to pH levels corresponding to those associated with the ammonia concentrations showed minimal effects of alkaline pH alone on oocyst viability. This study shows that environmentally relevant concentrations of free ammonia may significantly increase the inactivation of oocysts in ammonia-containing environments.     

Development and Preliminary Evaluation of a Loop-Mediated Isothermal Amplification Procedure for Sensitive Detection of Cryptosporidium Oocysts in Fecal and Water Samples

A loop-mediated isothermal amplification (LAMP) procedure for the detection of Cryptosporidium in environmental and fecal samples was developed and evaluated. This is the first demonstration of LAMP applied to detection of Cryptosporidium. Due to its specificity and simplicity, the method could become a useful diagnostic tool for epidemiologic studies of Cryptosporidium presence.     

Recovery and Enumeration of Cryptosporidium parvum from Animal Fecal Matrices

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 10² 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.     

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.     

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts

Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 μg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 μg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 μg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.     

Effects of Preservatives on Viability of Cryptosporidium parvum Oocysts

Potassium dichromate and formalin reduced the viability of Cryptosporidium parvum oocysts as assessed by inclusion or exclusion of 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) and excystation. Some formalin-treated oocysts containing dead sporozoites excluded PI; that this fluorogenic assay relies not solely upon exclusion of PI but also upon highlighting of sporozoite nuclei by DAPI is reiterated.     

Significance of Wall Structure,Macromolecular Composition,and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.Previous studies of the survival of Cryptosporidium parvum under natural and laboratory conditions have shown that the oocyst phase is a durable stage in the life cycle of this apicomplexan parasite and is crucial for parasite transmission. A major public health problem is the resistance of oocysts to chlorine at normal concentrations used in water treatment systems. Oocysts have the reputation of being tough, durable structures; however, they can be inactivated by many physical and chemical disinfectants, including UV radiation, ozone, ammonia, high temperature, desiccation, freezing, and exposure to extreme alkaline or acidic conditions (10, 11, 12, 17, 18, 22, 35). Low temperatures above freezing extend oocyst viability and infectivity for very long times (12, 18, 19, 20, 35). Environmental temperature is a major factor controlling oocyst survival (23, 24, 32). While there have been many studies documenting the significance of temperature for oocyst survival and the influence of temperature on stored energy reserve utilization has been recognized (see references 10 and 32 for reviews), the effects of temperature on the key oocyst wall structures and macromolecules have not been well investigated.While oocyst wall structure and macromolecular chemistry have been investigated in some detail (10, 14, 31, 33, 34, 41) and survival and transport in natural environments have been studied (5, 6, 8, 10, 23, 24), neither the underlying mechanisms by which oocysts resist environmental pressures nor the surface properties that control environmental transport have been well characterized (23, 24).In this study, we investigated details of the ultrastructure and chemical composition of the C. parvum oocyst wall with the aim of understanding the key physical and chemical properties of the oocyst wall that may confer environmental resistance and affect environmental transport.     

Occurrence of Cryptosporidium Oocysts and Giardia Cysts in Sewage in Norway

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of >20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and >80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.     

Low-Pressure UV Inactivation and DNA Repair Potential of Cryptosporidium parvum Oocysts

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm² (=30 J/m²), the reduction reached the cell culture assay detection limit of ~3 log₁₀. At UV doses of 1.2 and 3 mJ/cm², the log₁₀ reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.