Rotifers ingest oocysts of Cryptosporidium parvum

Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Eauchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.     

Gaseous disinfection of Cryptosporidium parvum oocysts.

Purified oocysts of Cryptosporidium parvum suspended in approximately 400 microliters of phosphate-buffered saline or deionized water in microcentrifuge tubes were exposed at 21 to 23 degrees C for 24 h to a saturated atmosphere of ammonia, carbon monoxide, ethylene oxide, formaldehyde, or methyl bromide gas. Controls were exposed to air. Oocysts in each tube were then rinsed and resuspended in fresh, deionized water, and 1 million oocysts exposed to each gas were orally administered to each of three to six neonatal BALB/c mice in replicate groups. Histologic sections of ileum, cecum, and colon tissues taken from each mouse 72 h after oral administration of oocysts were examined microscopically to determine if infection had been established. All 15 mice given oocysts exposed to carbon monoxide had numerous developmental stages of cryptosporidium in all three intestinal segments. Of 10 mice given oocysts exposed to formaldehyde, 6 had a few developmental stages of cryptosporidium in the ileum. No mice given oocysts exposed to ammonia, ethylene oxide, or methyl bromide were found to be infected. These findings indicate the efficacy of these low-molecular-weight gases (ammonia, ethylene oxide, and methyl bromide) as potential disinfectants for C. parvum oocysts where soil, rooms, buildings, tools, or instruments might be contaminated.     

Ultrastructural changes in Cryptosporidium parvum oocysts by gamma irradiation

Cryptosporidium parvum is known as one of the most highly resistant parasites to gamma irradiation. To morphologically have an insight on the radioresistance of this parasite, ultrastructural changes in C. parvum sporozoites were observed after gamma irradiation using various doses (1, 5, 10, and 25 kGy) following a range of post-irradiation incubation times (10 kGy for 6, 12, 24, 48, 72, and 96 hr). The ultrastructures of C. parvum oocysts changed remarkably after a 10-kGy irradiation. Nuclear membrane changes and degranulation of dense granules were observed with high doses over 10 kGy, and morphological changes in micronemes and rhoptries were observed with very high doses over 25 kGy. Oocyst walls were not affected by irradiation, whereas the internal structures of sporozoites degenerated completely 96 hr post-irradiation using a dose of 10 kGy. From this study, morphological evidence of radioresistance of C. parvum has been supplemented.     

Infectivity of Cryptosporidium parvum oocysts following cryopreservation.

This study was undertaken to investigate the cryopreservation of Cryptosporidium parvum oocysts. Oocysts purified from mouse feces were suspended in distilled water, 10% glycerin, and 2.5% potassium dichromate. They were stored at -20 C and -80 C for 2, 7, and 30 days, respectively. In addition to the purified oocysts, the feces of C. parvum-infected mice were preserved under the same conditions described above. Purified and fecal oocysts were thawed at 4 C, and their viability was assessed by a nucleic acid stain, excystation test, tissue culture infectivity test, and infectivity to immunosuppressed adult mice. Oocysts purified from fecal material prior to cryopreservation lost most of their viability and all of their infectivity for tissue culture and mice. However, when oocysts were cryopreserved in feces, between 11.7 and 34.0% were judged to be viable and retained their infectivity for mice when stored at -20 C (but not -80 C) for 2, 7, and 30 days. Clearly, fecal material provides a cryoprotective environment for C. parvum oocysts stored at -20 C for at least 30 days.     

Biofilms reduce solar disinfection of Cryptosporidium parvum oocysts

Solar radiation reduces Cryptosporidium infectivity. Biofilms grown from stream microbial assemblages inoculated with oocysts were exposed to solar radiation. The infectivity of oocysts attached at the biofilm surface and oocysts suspended in water was about half that of oocysts attached at the base of a 32-μm biofilm.     

Efficacy of UV irradiation in inactivating Cryptosporidium parvum 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-log(10) reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm(2) at 20 degrees C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm(2) for a 2-log(10) reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm(2). 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 degrees C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log(10) 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.     

Detection of viable Cryptosporidium parvum oocysts by PCR.

PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts.     

A comparison of enumeration techniques for Cryptosporidium parvum oocysts

A variety of methods have been used to enumerate Cryptosporidium parvum oocysts from source or drinking waters. The reliability of these counting methods varies, in part, with suspension density, sample purity, and other factors. Frequently, the method of determination of suspension density is not reported by authors. To confound the problem, each method of counting has large inherent variation. There is a relationship between suspension density, overall number of organisms counted, and counting mechanism accuracy that should be accounted for when selecting a counting mechanism. This study selected a maximum acceptable coefficient of variation (CV) to be 10%. A method was considered unreliable if this standard was not achieved. Flow cytometry achieved this standard at 486 oocysts/ml. Counting with a Coulter counter achieved this level of reliability at about 1,230 oocysts/ml. Neither chamber slides nor fluorescent antibody-stained well slides ever demonstrated less than 10% CV. However, estimates of the minimum required concentrations were 5,100 oocysts/ml and approximately 6,500 oocysts/ml, respectively. The hemacytometer provided counts accurate to a 10% CV at a concentration of at least 60,000 organisms/ml. Of the methods tested, flow cytometry provided the least amount of variability at low suspension densities.     

Inactivation of Cryptosporidium parvum oocysts in water using ultrasonic treatment

Ultrasound in a liquid phase cause mass and heat transfer across the liquid through cavitational processes which act as nanoreactors to generate unstable mechanical equilibrium. The effect of 1 MHz ultrasound on the inactivation of Cryptosporidium parvum was investigated. Continuous irradiation of ultrasound (20 min) increased temperature due to cavitational phenomena. Ultrasound irradiation of liquid containing C. parvum showed significant quantitative changes in pH, temperature and inactivation of C. parvum (102.7 oocysts killed/s) with a minimum energy consumption (0.05 oocysts/s).     

Infectious Cryptosporidium parvum Oocysts in Final Reclaimed Effluent

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.     

Evaluation of recovery of Cryptosporidium parvum oocysts using membrane filtration

Recovery of oocysts of Cryptosporidium parvum using 142 mm diameter 1.2 μm pore size acrylic copolymer membrane filters was evaluated. A mean recovery efficiency of 25.5% for oocyst concentrations of about 200 in 10 1 was achieved, making this method a simple and relatively efficient procedure compared with current standard methods.     

Contamination of river water by Cryptosporidium parvum oocysts in western Japan

In Japan, only a few rivers have been inspected for Cryptosporidium parvum contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for Cryptosporidium oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for Cryptosporidium oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the C. parvum found as the bovine or human type. C. parvum was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained C. parvum oocysts. The number of C. parvum oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the C. parvum detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with C. parvum oocysts, as reported in other countries.     

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.     

Infectivity of preserved Cryptosporidium parvum oocysts for immunosuppressed adult mice

Abstract The present study was undertaken to determine the infectivity of Cryptosporidium parvum oocysts for immunosup-pressed adult C57BL/6N mice after the oocysts had been stored from 1–48 months at 4°C in 2.5% potassium dichromate. All mice inoculated with oocysts 1–18 months old developed patent infections, while mice inoculated with older oocysts remained uninfected. The prepatent period was extended from 2 to 6 or 7 days as the storage time for oocysts increased. The finding that C. parvum oocysts remain infective for mice for at least 18 months offers important economic and time-saving advantages for investigators who frequently require large numbers of oocysts that must be painstakingly purified from calf manure.     

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.     

Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein

Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.     

Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.     

Survival of Cryptosporidium parvum oocysts in source separated human urine.

The survival of Cryptosporidium parvum in source separated urine was investigated as part of a broader study on microbial risks associated with the reuse of human urine for sustainable agriculture. A dye permeability assay and in vitro excystation were the primary methods used to assess viability. In the collected urine most of the nitrogen is present as ammonia and the pH is generally around 9. Parallel investigations were made in buffers to compare possible toxic effects of urine to actual pH effects. Oocysts in the untreated urine were inactivated below the detection limit (1/300) within 63 days. This inactivation rate was significantly higher (p < 0.01) than in urine adjusted to pH 5 or 7 according to the dye permeability assay. The corresponding difference between different pH values was not seen in buffers, suggesting that the antiprotozoan effect of urine was mediated by other factors besides pH. The Swedish practice of storing urine for six months before its use thus appears satisfactory for the inactivation of Cryptosporidium oocysts.     

A rapid method for producing highly purified Cryptosporidium parvum oocysts

Most procedures that have been described for purifying Cryptosporidium parvum oocysts are designed to either identify the parasites in clinical specimens or isolate oocysts from a small volume of feces from infected animals. The present study describes a rapid method for purifying high numbers of C. parvum oocysts from feces of infected calves that contains minimal contaminating fecal material and bacteria. The isolation method is based on differential flotation of C. parvum oocysts in NaCl, followed by ether extraction to solubilize lipids in calf feces. This procedure regularly yields > 10(9) purified C. parvum oocysts within 1-2 days of feces collection.