Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.     

Ultrastructure of Cryptosporidium parvum oocysts and excysting sporozoites as revealed by high resolution scanning electron microscopy

Cryptosporidium parvum oocysts isolated from calf feces were examined by scanning electron microscopy during excystation. Intact C. parvum oocysts were spheroid to ellipsoid, approximately equal to 3.5 X 4.0 micron, with length : width ratio = 1.17. The oocyst wall had a single suture at one pole, which spanned 1/3 to 1/2 the circumference of the oocyst. During excystation the suture dissolved, resulting in a slit-like opening, which the sporozoites used to exit the oocyst. Sporozoites were 3.8 X 0.6 micron and had a rough outer surface.     

In Vitro Excystation of Cryptosporidium baileyi from Chickens1

Release of sporozoites from the oocysts of Cryptosporidium baileyi is described from Nomarski interference-contrast microscopy. Just prior to excystation, the four sporozoites became motile and rearranged themselves within the oocyst. The sporozoites were then rapidly expelled through an opening that formed in the oocyst wall, and the residuum was either released or retained within the oocyst. Excysted sporozoites were crescent shaped and measured 5.0–9.0 μm × 1.0–1.6 μm (x?= 6.8 × 1.1 μm). Excystation occurred when sodium taurocholate or a mixture of trypsin and sodium taurocholate was present in the incubation medium. High levels of excystation occurred at 37° or 40°C, but excystation did not occur at 4°C. The ability of biles from two avian and two mammalian hosts to produce excystation of C. baileyi was also studied. After a 2-h incubation at 40°C, the percentages of excystation were 69.5% in goat bile, 45.0% in pig bile, 33.0% in chicken bile, and 34.5% in turkey bile.     

Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters

Aims: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters. Methods and Results: Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity. Conclusions: Solar UV can rapidly inactivate C. parvum in environmental waters. Significance and Impact of the Study: This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.     

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log₁₀ and 2.0 log₁₀ units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log₁₀ units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

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.     

Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.     

Environmental Inactivation of <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> Oocysts in Waste Stabilization Ponds

The survival of Cryptosporidium parvum oocysts in a waste stabilization pond system in northwestern Spain and the effects of sunlight and the depth and type of pond on oocyst viability were evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). All tested factors had significant effects (P < 0.01) over time on C. parvum oocyst viability. Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days exposure to sunlight conditions compared with dark conditions. The type of pond also caused a significant reduction in C. parvum oocyst viability (P < 0.01). Inactivation rates reflected that the facultative pond was the most aggressive environment for oocysts placed both at the surface (presence of sunlight) and at the bottom (absence of sunlight) of the pond, followed by the maturation pond and the anaerobic pond. The mean inactivation rates of oocysts in the ponds ranged from 0.0159 to 0.3025 day⁻¹.     

Association of Cryptosporidium parvum with Suspended Particles: Impact on Oocyst Sedimentation

The association of Cryptosporidium parvum oocysts with suspended particles can alter the oocysts' effective physical properties and influence their transport in aquatic systems. To assess this behavior, C. parvum oocysts were mixed with various suspended sediments under a variety of water chemical conditions, and the resulting settling of the oocysts was observed. Direct microscopic observations showed that oocysts attached to suspended sediments. Settling column and batch experiments demonstrated that oocysts are removed from suspension at a much higher rate when associated with sediments. The rate of oocyst sedimentation depended primarily on the type of sediment with which the oocysts were mixed. Changes in background water conditions had a relatively small impact on the extent of oocyst-particle association and the resulting oocyst deposition. We believe that the ubiquitous association of C. parvum oocysts with suspended particles enhances the sedimentation of oocysts in natural waters and that this interaction should generally be considered when predicting the migration of pathogens in the environment.     

Effect of various environmental factors on the viability of Cryptosporidium parvum oocysts

Aims: To evaluate individual and combined effects of temperature (4, 18 and 25°C), pH (7 and 10), ammonia (5 and 50 mg l^?1) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water. Methods and Results: The viability of oocysts was evaluated using the fluorogenic vital dyes assay (4′,6‐diamidino‐2‐phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P < 0·005). Exposure of oocysts to pH 10 for 6 days at 25°C reduced oocyst viability from ～80% to 51%. Similarly, the exposure of C. parvum oocysts to 5 mg NH₃ l^?1 and 50 mg NH₃ l^?1 for 4 days reduced their viability from between ～80% to 41·5% and 14·8%, respectively. Conclusions: The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low concentrations of ammonia, as commonly found in alga‐based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates. Significance and Impact of the Study: This study provides relevant data on the inactivation of C. parvum oocysts in alga‐based wastewater‐treatment systems in the northwest of Spain.     

Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice

Aim: To assess the efficiency of a medium‐pressure UV reactor under full‐scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. Methods and Results: Six/seven‐day‐old mice were administered orally 2–10 × 10⁴Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm^?2 UV irradiation of ingested oocysts resulted 7 days later in a 3·4–4·0 log₁₀ reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. Conclusion: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. Significance and Impact of the study: Present results suggest that in WTP conditions, a medium‐pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro‐organisms present in environmental waters.     

In Vitro Excystation of Caryospora simplex (Apicomplexa: Eimeriorina)1

In vitro excystation of sporozoites of the heteroxenous coccidian Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriorina) is described. Sporocysts freed mechanically from oocysts released a maximum of 51% of their sporozoites within 45 min at 25°C and a maximum of 74% within 20 min at 37°C when incubated in a 0.25% (w/v) trypsin–0.75% (w/v) sodium taurocholate (bile salt) excystation solution. At emergence from sporocysts, sporozoites were weakly motile then became highly active after about 2 min in excystation solution. Sporozoites within sporocysts exposed to bile salt only became highly motile within 25 min at 25°C and within 15 min at 37°C but did not excyst. When exposed only to trypsin at the above temperatures, the Stieda body dissolved; the substieda body remained intact, and the sporozoites exhibited only limited motility within sporocysts; only a few excysted. Intact, sporulated oocysts incubated at 25° or 37°C in 0.02 M cysteine-HC1 and a 50% CO₂ atmosphere for 18 h had no morphologic changes in the oocyst wall. Further incubation of these intact oocysts in excystation solution for 30 min at 37°C caused neither motility of sporozoites within sporocysts nor excystation. Grinding oocysts for 30 sec in a motor-driven, teflon-coated tissue grinder caused motility of some sporozoites within sporocysts but did not result in excystation.     

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M _L-cysteine HCl/0.2 M NaHCO₃ solution at 37 °C in 100% CO₂ atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.     

Efficacy of UV Irradiation in Inactivating Cryptosporidiumparvum Oocysts

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log₁₀ reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm² at 20°C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm² for a 2-log₁₀ reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm². Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log₁₀ reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.     

Induction of folds or sutures on the walls of Cryptosporidium parvum oocysts and their importance as a diagnostic feature.

The proportion of oocysts of Cryptosporidium parvum showing a fold on oocyst walls when incubated with either fluorescent monoclonal antibody or a surface-reactive fluorescent dye was increased by incubating suspensions of oocysts with dimethyl sulfoxide, sucrose, or Hanks' balanced salt solution. Further incubation of sucrose-incubated oocysts with water showed this to be a reversible phenomenon. Oocysts demonstrating this fold after incubation in dimethyl sulfoxide were of the same viability as control oocysts and followed the same excystation dynamics. Despite this fold having been previously described as a suture, we were unable to find any evidence that this pattern of fluorescence highlighted the same suture that has been described in ultrastructural studies. Furthermore, oocysts were observed in which this fold was not always continuous with the gape in the oocyst wall through which the sporozoites had emerged. We propose that this fluorescently highlighted region or fold should no longer be described as a suture and question its validity as a diagnostic feature. When environmental and other samples are being examined for the presence of C. parvum oocysts, objects of appropriate size, shape, and fluorescence which do not demonstrate a surface fold should not necessarily be excluded.     

Effect of Particles on the Recovery of Cryptosporidium Oocysts from Source Water Samples of Various Turbidities

Cryptosporidium parvum can be found in both source and drinking water and has been reported to cause serious waterborne outbreaks which threaten public health safety. The U.S. Environmental Protection Agency has developed method 1622 for detection of Cryptosporidium oocysts present in water. Method 1622 involves four key processing steps: filtration, immunomagnetic separation (IMS), fluorescent-antibody (FA) staining, and microscopic evaluation. The individual performance of each of these four steps was evaluated in this study. We found that the levels of recovery of C. parvum oocysts at the IMS-FA and FA staining stages were high, averaging more than 95%. In contrast, the level of recovery declined significantly, to 14.4%, when the filtration step was incorporated with tap water as a spiking medium. This observation suggested that a significant fraction of C. parvum oocysts was lost during the filtration step. When C. parvum oocysts were spiked into reclaimed water, tap water, microfiltration filtrate, and reservoir water, the highest mean level of recovery of (85.0% ± 5.2% [mean ± standard deviation]) was obtained for the relatively turbid reservoir water. Further studies indicated that it was the suspended particles present in the reservoir water that contributed to the enhanced C. parvum oocyst recovery. The levels of C. parvum oocyst recovery from spiked reservoir water with different turbidities indicated that particle size and concentration could affect oocyst recovery. Similar observations were also made when silica particles of different sizes and masses were added to seeded tap water. The optimal particle size was determined to be in the range from 5 to 40 μm, and the corresponding optimal concentration of suspended particles was 1.42 g for 10 liters of tap water.     

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.     

Use of Semiconductor Quantum Dots for Photostable Immunofluorescence Labeling of Cryptosporidium parvum

Cryptosporidium parvum is a waterborne pathogen that poses potential risk to drinking water consumers. The detection of Cryptosporidium oocysts, its transmissive stage, is used in the latest U.S. Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-Cryptosporidium sp. monoclonal antibody (MAb). However, FITC exhibits low resistance to photodegradation. This property will inevitably limit the detection accuracy after a short period of continuous illumination. In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection. In this study, QD605-streptavidin together with biotinylated MAb was used for C. parvum oocyst detection. The C. parvum oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above. The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 ± 501 oocysts (mean ± standard deviation) was determined to be 20 nM. The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (P > 0.126) when various C. parvum oocyst concentrations were used. The QD605 was highly photostable while the FITC intensity decreased to 19.5% ± 5.6% of its initial intensity after 5 min of continuous illumination. The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water. This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting C. parvum oocysts with the advantage of having a high photobleaching threshold.     

Impact of Zooplankton Grazing on the Excystation, Viability, and Infectivity of the Protozoan Pathogens Cryptosporidium parvum and Giardia lamblia

Very little is known about the ability of the zooplankton grazer Daphnia pulicaria to reduce populations of Giardia lamblia cysts and Cryptosporidium parvum oocysts in surface waters. The potential for D. pulicaria to act as a biological filter of C. parvum and G. lamblia was tested under three grazing pressures (one, two, or four D. pulicaria grazers per 66 ml). (Oo)cysts (1 × 10⁴ per 66 ml) were added to each grazing bottle along with the algal food Selenastrum capricornutum (6.6 × 10⁴ cells per 66 ml) to stimulate normal grazing. Bottles were rotated (2 rpm) to prevent settling of (oo)cysts and algae for 24 h (a light:dark cycle of 16 h:8 h) at 20°C. The impact of D. pulicaria grazing on (oo)cysts was assessed by (i) (oo)cyst clearance rates, (ii) (oo)cyst viability, (iii) (oo)cyst excystation, and (iv) oocyst infectivity in cell culture. Two D. pulicaria grazers significantly decreased the total number of C. parvum oocysts by 52% and G. lamblia cysts by 44%. Furthermore, two D. pulicaria grazers significantly decreased C. parvum excystation and infectivity by 5% and 87%, respectively. Two D. pulicaria grazers significantly decreased the viability of G. lamblia cysts by 52%, but analysis of G. lamblia excystation was confounded by observed mechanical disruption of the cysts after grazing. No mechanical disruption of the C. parvum oocysts was observed, presumably due to their smaller size. The data provide strong evidence that zooplankton grazers have the potential to substantially decrease the population of infectious C. parvum and G. lamblia in freshwater ecosystems.     

Cryptosporidium parvum Infection in SCID Mice Infected with Only One Oocyst: qPCR Assessment of Parasite Replication in Tissues and Development of Digestive Cancer

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10⁵ oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.