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 10⁷ 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° and 10°) 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° slope) to up to 10^4.5 oocysts from unvegetated soil (55-mm h⁻¹, 30-min event on 10° 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.     

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.     

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.     

Sensitivity of three grassland communities to simulated extreme temperature and rainfall events

Three grassland communities in New Zealand with differing climates and proportions of C3 and C4 species were subjected to one‐off extreme heating (eight hours at 52.5°C) and rainfall (the equivalent of 100 mm) events. A novel experimental technique using portable computer‐controlled chambers simulated the extreme heating events. The productive, moist C3/C4 community was the most sensitive to the extreme events in terms of short‐term community composition compared with a dry C3/C4 community or an exclusively C3 community. An extreme heating event caused the greatest change to plant community species abundance by favouring the expansion of C4 species relative to C3 species, shifting C4 species abundance from 43% up to 84% at the productive, moist site. This was observed both in the presence and absence of added water. In the absence of C4 species, heating reduced community productivity by over 60%. The short‐term shifts in the abundance of C3 and C4 species in response to the single extreme climatic events did not have persistent effects on community structure or on soil nitrogen one year later. There was no consistent relationship between diversity and stability of biomass production of these plant communities, and species functional identity was the most effective explanation for the observed shifts in biomass production. The presence of C4 species resulted in an increased stability of productivity after extreme climatic events, but resulted in greater overall shifts in community composition. The presence of C4 species may buffer grassland community productivity against an increased frequency of extreme heating events associated with future global climate change.     

模拟降雨下喀斯特坡耕地土壤养分输出机制

喀斯特区坡耕地水土及养分流失不仅是该区土地质量退化、土地生产力衰退主要原因,同时也是该区地下水质污染的重要因素。为揭示喀斯特坡耕地地表和地下二元空间结构下的土壤养分流失机制,以喀斯特坡耕地为研究对象,通过模拟其地表微地貌及地下孔(裂)隙构造特征,采用人工模拟降雨试验研究不同雨强下喀斯特坡耕地地表及地下水土及其氮、磷、钾流失特征。结果表明:(1)小雨强(50mm/h)和中雨强下(70mm/h),喀斯特坡耕地坡面产流主要以地下产流为主;大雨强下(90mm/h),地表径流高于地下径流;产沙方式则表现为由小雨的地表和地下产沙并重到中大雨强的地表产沙为主的一个转变过程。(2)在降雨侵蚀过程中,径流各养分输出浓度均表现出一定的初期冲刷效应,受土壤吸附作用影响,雨强对全钾(TK)和全氮(TN)的影响较全磷(TP)明显。(3)地表径流、地表泥沙和总泥沙各养分输出负荷均随雨强增大而增加,坡面径流泥沙总的TK输出负荷以泥沙为主,而TN和TP输出负荷则以径流为主;TP和TN在径流的输出负荷上以地下径流输出为主(其中TP地表负荷比在11.6%—46.2%,TN在7.0%—48.5%之间),而TK则以二者并重(地表负荷比在43.5%—57.0%之间);各养分在泥沙的输出负荷上则均以地表泥沙流失为主,其负荷比均在54.5%以上。研究结果可为喀斯特区坡耕地水土流失及养分流失的源头控制提供基本参数和科学依据。     

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 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.     

模拟降雨条件下生物结皮坡面产流产沙对雨强的响应

生物结皮是黄土丘陵区退耕地广泛存在的地被物.本研究通过人工模拟降雨试验分析了雨强对生物结皮坡面产流产沙的影响.结果表明: 生物结皮坡面产流产沙随降雨历时的延长先增加、10~18 min趋于平稳.相对于裸土坡面,生物结皮显著延长了坡面初始产流时间,抑制了坡面产流产沙,可降低21%~78%的坡面径流量和77%~95%的产沙量.雨强主要通过影响生物结皮坡面径流而影响其产沙.随着雨强的增加,生物结皮坡面产流产沙与雨强的相关性出现了由不显著相关向显著相关的转折,雨强＞1.5 mm·min^-1时,生物结皮坡面的减流减沙作用随着雨强的增加而降低.本研究结果为降雨条件下生物结皮坡面产流产沙过程的模型模拟奠定了基础.     

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.     

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.     

耕作对土壤入渗性能的影响

耕作影响降雨入渗、径流、土壤侵蚀过程。研究采用新型的坡地降雨条件下土壤入渗能力的径流-入渗-产流测量方法和仪器,野外观测宁夏南部山区坡耕地与撂荒地土壤入渗性能,揭示耕作对土壤入渗性能影响的规律。在坡度为20°、土壤表层含水量11.3%的坡地进行测量。采用的降雨强度为:20,40,56mm·h-1。结果表明,新型测量方法和仪器可以很好地测量坡面土壤入渗性能的全过程,并且具有省水、省工、操作方便的特点,且测量精度高。对比测量结果表明,坡耕地的初期土壤入渗性能在小雨强条件下略高于撂荒地的入渗性能,而在大雨强条件下则一直低于撂荒地的入渗性能。随雨强的增大坡耕地的入渗性能急剧减小,而撂荒地的入渗性能基本不受雨强变化的影响。坡耕地土壤的入渗性能(土壤结构)对降雨强度极为敏感,而撂荒地具有稳定的土壤结构和入渗性能。在3种雨强下,坡耕地的产流时间均比撂荒地提前。与撂荒地相比,各种雨强条件下坡耕地的稳定入渗率都较撂荒地明显降低。     

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.     

Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ·cm⁻² doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ·cm⁻². With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ·cm⁻² of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ·cm⁻² of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.     

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.