Survival Of Cryptosporidium Parvum Oocysts Research Articles

Persistence and survival of Cryptosporidium parvum oocysts on lamb's lettuce leaves during plant growth and in washing conditions of minimally-processed salads

Cryptosporidium is the causative agent of cryptosporidiosis, which results, among others, in profuse diarrhoea. Transmission to humans occurs via the faecal-oral route directly by contact with infected hosts or indirectly by waterborne or foodborne routes. For the latter, parasite transmission is closely linked to the oocyst's ability to persist and survive in food matrices. In this study, we evaluated the persistence and survival of Cryptosporidium oocysts in lamb's lettuce: i) during plant growth and ii) in conditions mimicking the industrial washing process applied in minimally-processed vegetables (MPV). Results show that oocysts persisted during the growth of lamb's lettuce, i.e. two months from the 2-leaf stage until the 8-leaf harvest time (−0.89 Log10 of oocysts). However, their survival decreased from as early as one week (−0.61 Log10), and only 6 % of oocysts remained infective at the time of harvest. The washing process had a limited effect on parasite load (<0.5 Log10) and no effect on survival; chlorination of washing water did not improve the efficiency (removal and inactivation) of the process. The ability of C. parvum to persist and survive throughout the food chain may drive its transmission to humans through MPV products. Appropriate management measures should be implemented at each operational level to limit contamination and ensure food safety of fresh produce.

Effect of Water Temperature on the Survival of Cryptosporidium parvum Oocysts by In vitro Excystation-Flow Cytometry Assay

Effect of Water Temperature on the Survival of Cryptosporidium parvum Oocysts by In vitro Excystation-Flow Cytometry Assay

Survival of Cryptosporidium parvum oocysts in the presence of hydrated lime

To investigate the effects of hydrated lime on the survival of Cryptosporidium oocysts, the percentage viability of oocysts was assessed using fluorescent in situ hybridisation. In the absence of lime...

Environmental Inactivation of Cryptosporidium parvum 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).

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic-thermophilic-mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of > or = 2 and > or = 5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Survival of Cryptosporidium parvum Oocysts after Prolonged Exposure to Still Natural Mineral Waters

The survival kinetics of purified Cryptosporidium parvum oocysts of both human and ovine origin, immersed in four still natural mineral waters (total dissolved salts ranging from 91 mg/liter to 430 mg/liter) and reverse osmosis water was assessed by inclusion or exclusion of the fluorogenic vital dyes 4′,6-diamidino-2-phenylindole and propidium iodide over a 12-week period. Semipermeable chambers were used to contain the oocysts while immersed in each mineral water type, permitting both intimate interactions between oocysts and matrices and straightforward sampling for viability assessments. The viability of both oocyst types, assessed at weekly intervals, remained unaltered after 12 weeks at 4°C, whereas a progressive decline in the viability of both oocyst isolates was observed when immersed in mineral waters at 20°C. At 20°C, approximately 30% of oocysts remained viable after 12 weeks incubation. Here, temperature was the major factor that adversely affected oocyst survival, although higher mineral content was also proportionally and significantly associated with this increased oocyst inactivation. The prolonged survival of oocysts at 4°C in our studies indicates that they could survive for prolonged periods of time in U.K. groundwaters (average temperature ~10°C) and thus represent a potential public health hazard if contamination of mineral water sources by viable oocysts were to occur.

Cryptosporidium parvum oocyst inactivation in three soil types at various temperatures and water potentials

The interaction between soil types, temperature, and soil water potential may have differential effects on the survival of Cryptosporidium parvum oocysts in the terrestrial environment. We examined the effects of three soil types (a silty clay loam, silt loam, and loamy sand), three temperatures (4, 20, and 30 °C), and three soil water potentials (−0.033, −0.5 and −1.5 MPa) on the inactivation kinetics of oocysts. Sentinel chambers were filled with air-dried and sieved soil, brought to the appropriate soil water potential, and inoculated with 2×10 6 freshly purified oocysts. The inoculated chambers were buried in the same bulk soil at the appropriate water potentials and incubated at one of the three temperatures. Triplicate chambers were removed from the bulk soil on days 0, 22, 43, 84 and 156. Sentinel oocysts were extracted, and assayed for potential infectivity by the dye permeability method. Oocysts suspended in sterile distilled water and incubated with the sentinel chambers were used as controls for the effect of temperature. The soil water potentials investigated did not affect oocyst inactivation at any temperature or with any of the three soil types. Rates of oocyst inactivation increased significantly between 4 and 20 °C, but not between 20 and 30 °C with the exception of oocysts incubated in the silty clay loam. Oocyst survival appeared to be significantly greater in the silt loam soil than in the two other soil types when incubated at 20 °C; and at 30 °C oocyst survival was significantly less in the silt clay loam than in the other two soil types. Rates of sentinel oocyst inactivation at all three soil water potentials were significantly lower than the control oocysts in water at the three test temperatures. Thus oocyst survival in soil was not affected by the water potentials between −0.033 and −1.5 MPa; it was affected by soil texture; but temperature appeared to be the factor most affecting oocyst survival. In the critical ambient range of temperature in temperate climates oocysts may survive for months in agricultural soil, and pose a threat to surface waters.

Survival of Cryptosporidium parvum oocysts recovered from experimentally contaminated oysters (Ostrea edulis) and clams (Tapes decussatus).

Samples of two species of shellfish that form part of the human food chain (the oyster Ostrea edulis and the marine clam Tapes decussatus) were experimentally contaminated with Cryptosporidium parvum oocysts. Changes in the viability of oocysts subsequently recovered from the shellfish were evaluated by means of an immunofluorescent antibody technique (IFAT) and inclusion/exclusion of the fluorogenic vital dye propidium iodide. There was a sharp decrease in oocyst viability during the first 4 days, with 15-25% viable oocysts remaining thereafter. In addition the infectivity of these oocysts at 10 and 31 days post-contamination was demonstrated using a suckling murine model.

Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels ( Mytilus galloprovincialis)

Transmission of infectious oocysts of Cryptosporidium parvum via surface- and drinking-water supplies has been reported and many surface waters flow into the sea, potentially causing runoff of animal-infected faeces. Eating raw mussels is a common practice in many countries, increasing the public's risk of acquiring enteric pathogens. The aims of the present study were to estimate how long C. parvum oocysts remain infectious in artificial seawater, to determine if the oocysts are retained in mussel tissues ( Mytilus galloprovincialis), and how long they maintain their infectivity. Oocysts were incubated in artificial seawater at 6–8°C under moderate oxygenation and the infectivity of oocysts was tested five times, over a 12 month period after incubation in seawater, in BALB/c mice. Each pup was inoculated per os with 10 5 oocysts and killed 5 days p.i. Oocysts remained infectious for 1 year. Forty mussels held in an aquarium containing artificial seawater filtered out more than 4×10 8 oocysts in a 24 h period. Oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the gut of mussels 7 and 14 days p.i. were observed to have infected mice. These results suggest that C. parvum oocysts can survive in seawater for at least 1 year and can be filtered out by benthic mussels, retaining their infectivity up to 14 days, so seawater and molluscs are a potential source of C. parvum infection for humans.

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 (NH(3)) 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 degrees 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. 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.

Survival of Cryptosporidium parvum oocysts under various environmental pressures.

The survival of various isolates of Cryptosporidium parvum oocysts under a range of environmental pressures including freezing, desiccation, and water treatment processes and in physical environments commonly associated with oocysts such as feces and various water types was monitored. Oocyst viability was assessed by in vitro excystation and by a viability assay based on the exclusion or inclusion of two fluorogenic vital dyes. Although desiccation was found to be lethal, a small proportion of oocysts were able to withstand exposure to temperatures as low as -22 degrees C. The water treatment processes investigated did not affect the survival of oocysts when pH was corrected. However, contact with lime, ferric sulfate, or alum had a significant impact on oocyst survival if the pH was not corrected. Oocysts demonstrated longevity in all water types investigated, including seawater, and when in contact with feces were considered to develop an enhanced impermeability to small molecules which might increase the robustness of the oocysts when exposed to environmental pressures.

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts.

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.