Infectivity Of Cryptosporidium Parvum Oocysts Research Articles

Efficacy of amine-based disinfectant KENO™COX on the infectivity of Cryptosporidium parvum oocysts

Cryptosporidium parvum is a zoonotic protozoan parasite that may cause severe neonatal diarrhoea or even mortality in newborn ruminants: its oocysts are extremely resistant to normal environmental conditions and to most common disinfectants. KENO™COX, a patent pending amine-based formula, was tested for its ability to inactivate C. parvum oocysts. The Daugschies assay (2002), a standardized assay for chemical disinfection initially described for Eimeria spp., was adapted for C. parvum oocysts. KENO™COX diluted in water at 2% and 3% concentration and incubated with oocyst suspensions for 2 h, allowed a significant reduction in viability, lysing 89% and 91% of oocysts respectively. Infectivity of the remaining C. parvum oocysts was assessed by inoculation to C57 Bl/6 neonatal mice. Each mouse received 2.5 μl of a suspension initially containing 500,000 oocysts before contact with KENO™COX. Six days post inoculation, the intestinal parasite load was significantly reduced by 97.5% with KENO™COX 2% compared to that of the mice inoculated with untreated parasites. KENO™COX 3% completely eliminated infectivity of oocysts. The number of oocysts remaining infectious in the inoculum treated with KENO™COX 2% was calculated from an inoculated dose–response curve: it was estimated at about 48.6 oocysts among the 500,000 oocysts initially treated corresponding to 99.99% of inhibition. These results demonstrate the high efficacy of KENO™COX against C. parvum oocysts. Combined with an appropriate method of cleaning, the application of KENO™COX may be a useful tool to reduce cryptosporidial infectious load on farm level.

Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples.

Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples after 6 weeks of cold storage, suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris-sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface.

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

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. Six/seven-day-old mice were administered orally 2-10x10(4)Cryptosporidium 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. Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. 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.

Transport, fate, and infectivity of Cryptosporidium parvum oocysts released from manure and leached through macroporous soil

A major mode of transmission of Cryptosporidium parvum, a widespread waterborne pathogen, is via contaminated drinking and recreational waters. Oocyst transport to surface water can occur by deposition of manure directly in the water or by wash off in surface runoff. Oocyst transport to groundwater is less straightforward and requires that the oocysts move through soil and bedrock to reach the water table. The purpose of this study was to determine the relative concentration and infectivity of C. parvum oocysts released from manure and leached through columns of undisturbed, macroporous karst soil. Modeling the fate of oocysts in this system over time can provide baseline data for evaluating real world events. Substantially more oocysts leached from undisturbed soil columns than disturbed soil columns. Oocyst survival studies using BALB/c neonatal suckling mice showed that about 85% of oocysts were infective at the beginning of leaching experiments. The oocyst infectivity decreased to about 20% after 12 weeks of leaching from soil columns maintained at 10°C. Cool (10°C) temperatures appear to increase survivability and maintain infectivity of many oocysts for 3 months or longer. Cool temperatures also appear to increase rates of release of oocysts from manure and leaching through soil. This study demonstrated that leaching is an important mechanism of oocyst transport in karst soils where infiltration capacities are high and long, continuous macropores exist. Karst groundwater systems might be especially vulnerable to contamination by leached oocysts, because of the prevalence of shallow soils and rapid groundwater movement. Oocysts leaching from soils into the epikarst could accumulate and remain viable for months until hydrological conditions are right for flushing the oocysts into the conduit flow system.

Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters

To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters. 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. Solar UV can rapidly inactivate C. parvum in environmental waters. 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.

Comparison of viability and infectivity of Cryptosporidium parvum oocysts stored in potassium dichromate solution and chlorinated tap water

The present study was undertaken to compare the viability and infectivity of Cryptosporidium parvum oocysts that had been stored for 1, 4, 7, 10, 13, 16, 20, 25 and 30 months at 4 °C in 2.5% potassium dichromate (Cr) or chlorinated tap water, respectively. An excystation protocol was performed in vitro to evaluate viability. One hundred and eighty female BABL/c mice were used to evaluate the infectivity of oocysts by investigating the prepatent period of C. parvum infection, the quantity of oocysts excreted, and the number of parasites that colonized the villi of the ileum. The results showed that C. parvum oocysts preserved in Cr for 1–16 months or in water for 1–13 months were capable of excystation in vitro and infection of mice. The excystation rates of oocysts and the prepatent periods in mice infected by oocysts stored in Cr and water were not significantly different ( p > 0.05), and there was a strong correlation between prepatent period and duration of oocyst storage (Cr: R 2 = 0.92 ; water: R 2 = 0.98). There were no significant differences in oocyst shedding from feces or parasitism of the terminal ilea of mice by Cryptosporidia between the two storage media ( p > 0.05). In conclusion, C. parvum oocysts may be stored at 4 °C in water instead of Cr for the purposes of laboratory research. However, the presence of viable C. parvum oocysts in water is a severe challenge to the drinking water treatment industry.

Pilot-scale evaluation of UV reactors' efficacy againstin vitroinfectivity ofCryptosporidium parvumoocysts

An experimental protocol was developed to assess the efficacy of two UV reactors (medium-pressure UVaster), and a low-pressure reactor) on the infectivity of Cryptosporidium parvum oocysts under conditions mimicking small- or medium-size water distribution units. The protocol included purification of large amounts of viable oocysts from experimentally infected calf feces, pilot spiking, sample concentration and purification after UV radiation, oocyst quantification and in vitro evaluation of oocyst infectivity on HCT-8 cells. Water samples were collected at intervals upstream and downstream from the UV reactor after spiking. Oocysts were concentrated by centrifugation, purified by immunomagnetic capture and quantified using laser-scanning cytometry. An enhanced in vitro infectivity test on HCT-8 cells was developed, where oocysts were pretreated in order to obtain maximized in vitro infectivity, and infectious foci were enumerated after immunofluorescence staining after 3 days of culture. This method was superior to viability measured by excystation for assessing oocyst infectivity. The infectivity rate of untreated oocysts ranged between 9% and 30% in replicate experiments. The method allowed us to determine inactivation rates >4.92 (log) with UVaster and >4.82 with the LP reactor after exposition of oocysts to an effective dose of 400 J m(-2) at flow rates of 15 and 42 m(3) h(-1), respectively.

銅管による<I>Cryptosporidium parvum</I>オーシストの感染性不活化

We studied whether the infectivity of Cryptosporidium parvum oocysts for suckling mice could be inactivated by copper tubing or by other types of tubing used to construct water distribution systems, including stainless steel, rigid polyvinyl chloride (PVC), PVC-lined steel, polyethylene (PE), cross-linked PE, and polybutene (PB), using glass tubing as the control. Oocysts were incubated in each tubings for 24 hours. The extent of inactivation of infectious oocysts by copper tubing was -1.303 log, which significantly inactivated of infectivity. In contrast, other types of tubing had no significant effect on some oocyst infectivity, although PB did show a maximum inactivation of -0.313 log. 25% of oocysts showed degeneration morphologically after passing through copper tubing, while 0.3% to 1.8% showed degeneration after passing through other tubing. Significant inactivation of infectious oocysts was not caused by water in which copper tubing had been let stand for 24 hours, although it had a cupric ion (Cu2+) concentration of 2.4 mg/L. The direct contact of oocysts with copper surface resulted in a decrease in the recovery percentage of oocysts and generation of hydrogen peroxide (0.5 mg/L) after 24 h of incubation. The percentage of degenerating oocysts was 29%. Such cryptosporidicidal effects of the copper surface on oocysts were completely inhibited by overlaying the surface with a Millipore filter before adding oocysts and incubating oocysts in the presence of catalase, an antioxidant enzyme. These findings suggest that copper tubing inactivates infectious C. parvum oocysts cytotoxically which may be due to oxygen radicals generated by the interaction between Cu2+ and hydrogen peroxide on the tubing surface.

Inactivation of Cryptosporidium oocysts and Giardia cysts by ultraviolet light in the presence of natural particulate matter

Recent studies have shown that ultraviolet (UV) radiation reduces the infectivity of Cryptosporidium parvum oocysts and Giardia spp. cysts. The objective of this work was to further examine UV inactivation of C. parvum oocysts and G. muris cysts in the presence of suspended particulate matter. Naturally occurring particulate matter consisting of a heterogeneous mixture of biological and non-biological particles, mainly in the 5–25 μm size range, was concentrated from a lake. Different aliquots of this material were mixed with purified oocyst and cyst preparations in aqueous suspensions, and the suspensions were exposed to UV fluences of 5 and 40 mJ/cm 2 from a medium-pressure mercury arc lamp using a collimated beam apparatus. Parasite inactivation was determined using mouse infectivity assays. Addition of particulate matter was correlated with statistically significant reductions in C. parvum and G. muris inactivation of 0.8 log 10 and 0.4 log 10 , respectively, even after the fluence was adjusted for increased absorbance due to the presence of the particles. The corresponding increases in turbidity were 0.3–20 NTU for C. parvum and 7.5–20 NTU for G. muris . The magnitude of the effect was a function of the final suspension turbidity but was independent of fluence. In this study, the effect of particles on parasite inactivation at turbidity values of less than 10 NTU was small and difficult to measure, and would likely be unimportant in practice. Although the mechanism for this reduction was not determined, the results suggest that the particulate matter present in natural surface waters may interact with oocysts and cysts and thereby result in a reduction in UV inactivation over and above that attributable to simple absorbance. The effect of turbidity on inactivation measured in this work may not necessarily apply generally since the characteristics of the particles that compose turbidity may differ considerably between water sources.

Evaluation of two commercial disinfectants on the viability and infectivity of Cryptosporidium parvum oocysts

Cryptosporidiosis is mainly a problem in neonatal ruminants. Not only do Cryptosporidium spp. spread ubiquitously in our environment, but the protozoa are highly resistant to harsh environmental conditions and disinfectants, and a control measure is urgently required. This study investigated the potential biocidal activity on Cryptosporidium parvum oocysts of two commercial disinfectants developed originally to be used in farms and food-processing industries. The products, containing formaldehyde and hydrogen peroxide respectively, both had some anticryptosporidial effects. The viability and infectivity of purified C. parvum oocysts exposed to both disinfectants at different concentrations and exposure times were evaluated by inclusion or exclusion of vital dye (propidium iodide), use of an excystation technique and infection of suckling mice. Viability assays showed a decrease in oocyst viability associated with an increase in exposure time for each of the concentrations used. The intensity of infection in neonatal mice was significantly lower ( P < 0.05) than in the control litters.

The effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum on beef surfaces

The aim of this research was to examine the effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum oocysts attached to a beef surface. This study examined the effects of heat treatment (60 or 75 degrees C) on the viability of C. parvum oocysts inoculated onto the surface of beef muscle estimated by vital dye assay. The infectivity of the oocysts was assessed against monolayers of HCT-8 cells. At 60 degrees C viability of the oocysts decreased from 100% at T0 to 64.2% at T60. At 75 degrees C the viability of the oocysts decreased from 100% at T0 to 53.7% at T15 and finally to 11.2% at T60. Oocysts were rendered noninfective against monolayers of HCT-8 cells following treatments of 60 degrees C/45 s and 75 degrees C/20 s. The washing of carcasses with hot water and standard thermal treatments is sufficient to kill C. parvum on beef. This study found that relatively mild heat, currently used to decontaminate and heat treat beef carcasses and to cook meat products, is capable of inactivating C. parvum.

Time and Temperature Effects on the Viability and Infectivity of Cryptosporidium parvum Oocysts in Chlorinated Tap Water

The authors compared the viability and infectivity of Cryptosporidium parvum oocysts in chlorinated tap water at various storage durations (i.e., 2 wk, 4 wk, 6 wk, or 8 wk) and at 2 cool temperatures (i.e., 10 degrees C and 4 degrees C), using in vitro (excystation) and in vivo (suckling mouse) methods. After 8 wk, mean oocyst excystation decreased to 33.4% and 26.7% at 10 degrees C and 4 degrees C, respectively. Suckling mice infectivity was higher after storage at 10 degrees C than after storage at 4 degrees C. These data suggest that Cryptosporidium parvum oocysts can survive and remain infectious for 8 wk in cool chlorinated tap water.

Viability and infectivity of Cryptosporidium parvum oocysts detected in river water in Hokkaido, Japan.

The viability and infectivity of Cryptosporidium parvum (C. parvum) oocysts, detected in water samples collected from river water in Hokkaido, were investigated using Severe Combined Immunodeficient (SCID) mice. The water samples collected from September 27 through October 10, 2001 by filtration using Cuno cartridge filters were purified and concentrated by the discontinuous centrifugal flotation method. From 1.2 x 10 (5) liters of the raw river water, approximately 2 x 10(4) oocysts were obtained and designated as Hokkaido river water 1 isolate (HRW-1). Oocyst identification was carried out using microscopic and immunological methods. Six 8-week-old female SCID mice were each inoculated orally with 1 x 10 (3) oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Oocysts were then maintained by sub-inoculation into SCID mice every 3 months. Infectivity was evaluated by making comparisons with two known C. parvum stocks, HNJ-1 and TK-1, which were bovine genotypes detected in fecal samples from a cryptosporidiosis patient and young cattle raised in Tokachi, Hokkaido respectively. The oocyst genotypes were determined from a small subunit ribosomal RNA (SSU-rRNA) gene by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences were observed in the average number of oocysts per gram of feces (OPG) in any of the isolates. Our data indicates that the C. parvum oocysts detected in the sampled river water were of C. parvum genotype 2. Moreover, our data on the continued isolation, detection and identification of the C. parvum isolates is consistent with the available epidemiological data for the Tokachi area.

Effect of the mixed-oxidant solution on infectivity of Cryptosporidium parvum oocysts in a neonatal mouse model

Cryptosporidium parvum oocysts were exposed to the mixed-oxidant solution, which was electrochemically generated by Miox Water Disinfection Unit, and sodium hypochlorite in phosphate buffered saline (PBS, pH 7.2) or biologically treated wastewater at 25 degrees by using concentrations of residual chlorine of up to 5 mg/l and contact times of up to 8 h. The effect of two disinfectants on infectivity of the oocysts in a neonatal murine model was comparatively evaluated by determining the total number of oocysts recovered from the intestine. Exposure to the mixed-oxidant solution at 2 and 5 mg/l (residual chlorine) yielded a significant inactivation of infectivity in the dose- and exposure time-dependent manner, while exposure to 5 mg/l (residual chlorine) of sodium hypochlorite for contact times of up to 4 h produced no measurable inactivation of infectivity. Morphological examination also revealed a picture of degenerating oocysts after exposure to 5 mg/l (residual chlorine) of the mixed-oxidant solution, but not with sodium hypochlorite. When the oocysts were exposed to either biologically treated wastewater--or PBS-diluted the mixed-oxidant solution at 5 mg/l (residual chlorine) for 4 h, the disinfectants produced a significant inactivation of infectious oocysts. The decrease number of the oocysts was 0.8 log 10 in the former and 2.1 log 10 in the latter. These results demonstrate that the mixed-oxidant solution may be a useful disinfectant against Cryptosporidium oocysts, but appropriate applications need to be validated.

Influence of temperature on Cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay.

Cryptosporidium parvum oocysts were stored in 1-ml aliquots of filtered river water at -20, 4, 10, and 21-23 C in the dark. Oocysts were also added to filter-sterilized river water samples and stored at 21-23 C. The infectivity of oocysts stored under different conditions was assayed at weekly intervals through infection of human adenocarcinoma ileocecal (HCT-8) cell monolayers. Wells containing between 10 and 100 foci of infection were enumerated by immunofluorescent microscopy, and the number of infective oocysts was calculated. No infectious oocysts were detected after 1 wk at -20 C. The number of infective oocysts stored at 4 C decreased 5-fold, and the number of those stored at 10 C decreased 2.5-fold after 14 wk. The infectivity of oocysts stored in potassium dichromate (positive control) at 4 C decreased 2-fold over 14 wk. The number of infective oocysts in filter-sterilized and non-filter-sterilized river water stored at 21-23 C decreased by 3.3 and 2.6 log units, respectively, over 12 wk, and no foci of infection were detected at 14 wk. The results show that as temperature increased from 4 to 23 C, the duration of oocyst infectivity decreased.

Influence of Temperature on Cryptosporidium parvum Oocyst Infectivity in River Water Samples as Detected by Tissue Culture Assay

Influence of Temperature on Cryptosporidium parvum Oocyst Infectivity in River Water Samples as Detected by Tissue Culture Assay

Efficacy of Common Laboratory Disinfectants on the Infectivity of Cryptosporidium parvum Oocysts in Cell Culture

Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts.

ELISAを用いた&lt;i&gt;Cryptosporidium parvum&lt;/i&gt;オーシストの生育活性評価法と紫外線による不活化の検討

Two ELISA methods were investigated for the quantitative detection of viability and infectivity of Cryptosporidium parvum oocysts. One is to detect the sporozoites attached to the cell surface, and another is to detect the oocysts developing in host cell. It was thought that the former method, named “Fixed-cell” ELISA, could assess the viability of oocysts, and the latter method, named “Living-cell” ELISA, could assess the infectivity of oocysts. In both ELISA systems, the resulting optical density was related to the number of oocysts inoculated. Inactivation of oocyst by ultraviolet (UV) irradiation was evaluated using these two ELISA systems. One-log reduction in infectivity of oocysts was achieved by 10 mW·s·cm-2 of UV irradiation. At this UV dose, no reduction in viability determined by “Fixed-cell” ELISA was observed.

Improving the Rate of Infectivity of Cryptosporidium parvum Oocysts in Cell Culture Using Centrifugation

Improving the Rate of Infectivity of Cryptosporidium parvum Oocysts in Cell Culture Using Centrifugation

Infectivity of Cryptosporidium parvum Oocysts Following Cryopreservation

Infectivity of Cryptosporidium parvum Oocysts Following Cryopreservation