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PubMed1980

Effects of storage time and temperature on amylopectin levels and oocyst production of Eimeria meleagrimitis oocysts.

Eimeria meleagrimitis oocysts were stored at 4, 22, 32, or 41.5 degrees C for up to 1 year. Decreases in amylopectin levels (measured as glucose) and viability (measured as oocyst production and mortality in turkeys) of the oocysts were generally related to the length of time in storage and the storage temperature. Oocysts assayed immediately after harvest contained 58.29 +/- 0.75 micrograms of glucose/10(6) oocysts. When the oocysts were stored at 4 degree C for 162 days, the amylopectin level decreased to 65% of the original level. In oocysts stored at 22, 32 and 41.5 degrees C, amylopectin declined to approximately 20% within 162, 76, and 41 days, respectively. Oocysts stored at 4 degree C for 1 year produced more oocysts in turkeys than the original fresh isolate, but caused no mortality. Oocyst production from oocysts stored at 22 and 32 degrees C decreased gradually until, after 9 and 7 months respectively, no patent infections were produced. Oocyst production from oocysts stored at 41.5 degree C markedly reduced within 1 month and was not detected after 4 months.

Amylopectin↗
PubMed2002

Improved production of Neospora caninum oocysts, cyclical oral transmission between dogs and cattle, and in vitro isolation from oocysts.

Scarce information is available about Neospora caninum oocysts and sporozoites, in part because only small numbers of oocysts have typically been produced by experimentally infected dogs. We hypothesized that I reason for low experimental production of oocysts is that dogs have been fed tissues from experimentally infected mice instead of tissues from cattle (which are natural intermediate hosts of N. caninum). In this study, 9 dogs were fed tissues from N. caninum-infected calves, and oocyst production was compared with 6 dogs that were fed infected mouse carcasses. The number of oocysts produced by dogs that ingested infected calf tissues (mean = 160,700) was significantly greater (P = 0.03) than the number of oocysts shed by dogs that ingested infected mice (mean = 5,400). The second goal of our experiment was to demonstrate cyclical oral transmission of N. caninum between dogs and cattle. As few as 300 oocysts were used to successfully infect calves, and tissues from these calves induced patent infections in 2 of 3 dogs; oocysts from I of these dogs were administered to another calf, and tissues from this calf subsequently induced a third dog to shed oocysts. Oocysts were confirmed to be N. caninum using a species-specific polymerase chain reaction technique. In addition, sporulated oocysts were used to recover N. caninum in vitro after digestion in an acid-pepsin solution and inoculation of cell monolayers.

Animals↗
PubMed1996

Killing of Toxoplasma gondii oocysts by irradiation and protective immunity induced by vaccination with irradiated oocysts.

Toxoplasma gondii oocysts are highly resistant to environmental influences. To study the effect of alpha-irradiation on the viability of T. gondii oocysts, 1 million sporulated oocysts of the VEG strain were irradiated to absorbed doses of 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, or 0.50 kGy at 5 C with a 137Cs gamma-irradiation source. Treated oocysts were bioassayed for viable T. gondii in mice. Viable T. gondii was not found in brains of mice 2 mo after oral inoculation with oocysts irradiated at levels > or = 0.25 kGy. Therefore, alpha-irradiation is an effective means of killing T. gondii oocysts. Mice inoculated orally with oocysts irradiated at 0.20 and 0.40 kGy were partially protected when challenged orally with lethal doses of nonirradiated oocysts.

Animals↗
PubMed2005

Immunomagnetic separation of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall.

Recent outbreaks of waterborne toxoplasmosis have stimulated the development of sensitive methods to detect Toxoplasma gondii oocysts in samples suspected to be contaminated. The immunomagnetic separation (IMS) have been standardised to detect waterborne protozoa, but it did not exist for Toxoplasma oocysts. In this study, we describe two monoclonal antibodies (mAbs 3G4 and 4B6) produced against the oocyst wall, and the incorporation of mAb 3G4 in an IMS procedure. We found that an indirect IMS method gave better mean recoveries than a direct one (69.4% and 25.2%, respectively). Dissociation of oocyst_magnetic bead complexes was greatly improved by using a 2% aqueous H2SO4 solution instead of a 0.1 N HCl solution (82.8% and 17.4%, respectively). With these parameters, mean recoveries of less than 1000 oocysts ranged from 44.6% to 82.9%, depending on incubating temperature and buffer. Age of oocysts (1 or 12 months old) does not influence IMS performances. Results of this study indicate that the described IMS is an efficient technique to recover Toxoplasma oocysts.

Animals↗
PubMed2006

Cryptosporidium oocyst surface macromolecules significantly hinder oocyst attachment.

The role Cryptosporidium parvum oocyst surface macromolecules play in controlling oocyst adhesion (deposition) kinetics to quartz surfaces has been investigated utilizing a radial stagnation point flow system. Deposition kinetics and corresponding attachment efficiencies of viable oocysts were compared with those after treatment with a digestive enzyme (proteinase K) to cleave these surface macromolecules. Low deposition rates were observed with viable oocysts over the entire range of ionic strengths (KCl) investigated, even at ionic strengths as high as 100 mM where the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloidal stability predicts the absence of an electrostatic energy barrier. "Electrosteric" repulsion between the oocyst surface macromolecules and the quartz surface is surmised to cause these low deposition rates and attachment efficiencies. However, after removal of these surface macromolecules by the digestive enzyme, increased attachment efficiencies were observed over the entire range of ionic strengths. This significant increase in the deposition kinetics was seen despite the oocysts having a more negative zeta potential following the removal of the surface macromolecules. After treatment with proteinase K, the oocysts no longer experienced electrosteric repulsive forces, and their deposition kinetics followed the general behavior predicted by DLVO theory.

Animals↗
PubMed1997

Oocyst-induced murine toxoplasmosis: life cycle, pathogenicity, and stage conversion in mice fed Toxoplasma gondii oocysts.

The development of sporozoites to tachyzoites and bradyzoites was studied in mice after feeding 1-7.5 x 10(7) Toxoplasma gondii oocysts. Within 2 hr after inoculation (HAI), sporozoites had excysted and penetrated the small intestinal epithelium. At 2 HAI, most sporozoites were in surface epithelial cells and in the lamina propria of the ileum, and by 8 HAI, T. gondii was also seen in mesenteric lymph nodes. At 12 HAI, sporozoites had divided into 2 tachyzoites in the lamina propria of the small intestine. By 48 HAI, there was a profuse growth of tachyzoites in the intestine and mesenteric lymph nodes of mice fed 7.5 x 10(7) oocysts. Parasites had disseminated via the blood and lymph to other organs by 4 days after inoculation (DAI). Toxoplasma gondii was first isolated from peripheral blood at 4 HAI. Tissue cysts were visible histologically in the brain at 8 DAI. By using immunohistochemical staining with anti-bradyzoite-specific (BAG-5 antigen) serum, BAG-5-positive organisms were first seen at 5 DAI in the intestine and at 8 DAI in the brain. Using the bioassay in cats, bradyzoites were first detected in mouse tissues between 6 and 7 DAI, and they were found in intestines before they were found in the brain. Cats fed murine tissues containing bradyzoites shed oocysts in their feces with a short (< 10 days) prepatent period, whereas cats fed tissues containing tachyzoites did not shed oocysts within 3 wk. Using a pepsin-digestion procedure and mouse bioassay, bradyzoites were first detected in brain tissue at 7 DAI and in many organs of mice at 51 and 151 DAI. Individual bradyzoites, small and large tissue cysts, and tachyzoites were seen in the brains of mice at 87 and 236 DAI.

Animals↗
PubMed1988

On the status of Eimeria nieschulzi oocysts embedded in resin eleven years ago: a permanent method for preserving coccidian oocysts.

Sporulated oocysts of Eimeria nieschulzi that were fixed and mounted on glass slides in polymerized resin in 1976 are examined. Size, shape, and integrity of oocysts and sporocysts are compared to similar observations we made in 1977 and reported in 1978 (Journal of Parasitology 64: 163-164). Our conclusion is that the methods we reported on in 1978 provide one opportunity to produce permanent specimens of sporulated oocysts that could be made available for deposit in nationally accredited museums.

Animals↗
PubMed2001

Decrease in Cryptosporidium parvum oocyst infectivity in vitro by using the membrane filter dissolution method for recovering oocysts from water samples.

Exposure of Cryptosporidium parvum oocysts to solutions used for cellulose acetate membrane (CAM) dissolution filtration reduced their infectivity in HCT-8 cells. Ethanol (95% [vol/vol] and 70% [vol/vol]) alone and short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.

Animals↗
PubMed1997

Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.

The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in distilled water showed that the shift in oocyst populations from impermeable to partially permeable to fully permeable was accelerated at temperatures above 4 degrees C. This sequence of oocyst permeability changes was taken as an indicator of the oocyst inactivation pathway. Using the dye permeability results, inactivation rates of oocysts in two fecal pools stored in the dark at 4 degrees C for 410 and 259 days were estimated to be 0.0040 and 0.0056 oocyst day-1, respectively. The excystation assay gave similar inactivation rates of 0.0046 and 0.0079 oocyst day-1. These results demonstrate the utility of the dye permeability assay as an indicator of potential viability and infectivity of oocysts, especially when combined with improved microscopic methods for detection of oocysts in soil, turbid water, and sediments.

Animals↗
PubMed1996

Infectivity and pathogenicity of Toxoplasma gondii oocysts for cats.

Toxoplasma gondii oocysts are highly infective to intermediate hosts including humans, pigs, and mice, but are considered less infective for cats, the definitive host. To determine infectivity of T. gondii oocysts for cats, 20 2- to 3-mo-old T. gondii-free cats in groups of 4 were fed graded doses of oocysts estimated to have 1, 10, 100, 1,000, or 10,000 mouse infective oocysts of the VEG strain of T. gondii. Feces of cats were examined for at least 35 days after feeding oocysts. All cats were killed, necropsied, their sera were tested for T. gondii antibodies, and tissues were bioassayed in mice. Three of the 4 cats fed 10,000 oocysts, 3 of the 4 cats fed 1,000 oocysts, and 2 of the 4 cats each fed 100 oocysts shed 7.3-162 million T. gondii oocysts in their feces, with a prepatent period of 18-44 days. Based on bioassay and antibody production, all 4 cats fed 10,000 oocysts, 3 of 4 cats fed 1,000 oocysts, 2 of 4 cats fed 100 oocysts, and 0 of 8 cats fed 1 or 10 oocysts acquired T. gondii infection. Antibodies to T. gondii were detected by the modified agglutination test in all 9 bioassay-proven T. gondii-infected cats and in none of the 11 cats without demonstrable T. gondii. In a series of other experiments, the age of the cat at the time of oocyst feeding and the administration of corticosteroids were found to have no influence on the prepatent periods after ingestion of oocysts. A review of published and unpublished data indicated that the minimum prepatent period to shedding of oocysts after the ingestion of oocysts by cats is 18 days.

Age Factors↗
PubMed2003

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.

Animals↗
PubMed1997

Infectivity of Cryptosporidium parvum oocysts is retained upon intestinal passage through a migratory water-fowl species (Canada goose, Branta canadensis).

Five Cryptosporidium-free Canada geese (Branta canadensis) were individually orally dosed with 3.5 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. After intestinal passage, inoculum-derived oocysts extracted from goose faeces established severe infection in 14 neonatal BALB/c mice (inoculum dose 2.5 x 10(5)/mouse). The inoculum-derived oocysts were detected in goose faeces up to 9 days post-inoculation (PI); the number of intact oocysts and oocyst shells shed during the first 3 days PI was significantly higher than for the remaining 6 days PI (P < 0.01). Based on acid-fast stained air-dried direct wet smears, 62% of the oocysts in goose faeces were intact (oocyst shells) constituted 38%) and conformed to morphological features of viable and infectious inoculum oocysts. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the faeces-recovered oocysts (majority 3+ to 4+). The dynamics of oocyst shedding showed that overall, the birds released a significantly higher number of intact oocysts than oocyst (P < 0.01). Retention of the viability and infectivity of C. parvum oocysts following intestinal passage through a migratory water-fowl species has serious epidemiological implications. Water-fowl can serve as mechanical vectors for the water-borne oocysts and can contaminate surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to water-shed management practices, water-shed protection programme officials should consider water-fowl as a potential factor enhancing contamination of the source water with Cryptosporidium.

Animals↗
PubMed1991

Fixing coccidian oocysts is not an adequate solution to the problem of preserving protozoan type material.

Fresh (36 days old) sporulated oocysts of Eimeria nieschulzi were divided into 7 groups. Control oocysts were maintained at 23 C in 2% aqueous (w/v) K2Cr2O7. The 6 experimental groups were mixed with either Bouin's solution, 10% aqueous (v/v) buffered formalin, Karnovsky's solution, glutaraldehyde, paraformaldehyde, or 70% aqueous (v/v) ethanol (EtOH). After 115 days, oocysts from all 7 groups were examined under oil immersion to determine the effect of fixation on their structural integrity. The parameters examined were lengths and widths of oocysts and sporocysts, percent sporulation (%S), and percent crenation (%C) of oocysts and sporocysts. The highest destruction (%S and %C) occurred in oocysts exposed to glutaraldehyde and Karnovsky's fixatives where 100% of both oocysts and sporocysts crenated and only 8% and 48%, respectively, remained sporulated. Of the oocysts in paraformaldehyde, 93% remained sporulated, but 95%of these oocysts and 100% of the sporocyst crenated. In Bouin's solution, 75% of the oocysts were intact structurally, but of these, only 60% were still sporulated with 70% of their sporocysts crenated. Oocysts preserved in 70% EtOH were 80% intact and 70% remained sporulated, but nearly 60% of their sporocysts collapsed even though the oocyst walls were intact. Oocysts preserved in 10% buffered formalin maintained structural integrity but had lower numbers of sporulated oocysts (84%) and greater numbers of crenated oocysts (18%) than control oocysts maintained in the dichromate solution (95% and 0%, respectively).

Acetates↗
PubMed2005

Settling behavior of unpurified Cryptosporidium oocysts in laboratory settling columns.

The settling behavior of fresh and aged unpurified oocysts was examined in settling column suspensions with varied ionic strengths and concentrations of calcium and magnesium. Independent measurements of the size and density of unpurified oocysts were performed to determine a theoretical settling velocity for the test populations. Viability of the oocysts was assessed using a dye permeability assay. Latex microspheres were included to provide a standard by which to assess the settling conditions in the columns. Mean settling velocities for viable oocysts measured in this work were faster than predicted and faster than measured for purified oocysts in other work: 1.31 (+/-0.21) microm/s for viable oocysts from populations having a low percentage of viable oocysts and 1.05 (+/-0.20) microm/s for viable oocysts from populations with a high percentage of viable oocysts. Results were attributed to the higher than previously reported densities measured for oocysts in this study and the presence of fecal material, which allowed opportunity for particle agglomeration. Settling velocity of oocysts was significantly related to the viability of the population, particle concentration, ionic strength, and presence of calcium and magnesium in the suspending medium. Behavior of the latex microspheres was not entirely predictive of the behavior of the oocysts under the test conditions. Viable oocysts may have a greater probability of settling than previously assumed; however, nonviable, and especially nonintact, oocysts have the potential to be significantly transported in water. This work underscores the importance of assessing the viability of oocysts to predict their response to environmental and experimental conditions.

Animals↗
PubMed1970

The Toxoplasma gondii oocyst from cat feces.

Coccidian oocysts resembling those of Isospora bigemina were excreted by cats fed Toxoplasma. In order to identify these oocysts with Toxoplasma infectivity a number of critical comparisons were made. The appearance of oocysts and Toxoplasma infectivity was simultaneous in the feces of 23 of 24 adult cats, 3-5 days after feeding of Toxoplasma cysts; in the feces of 4 out of 9 cats, 7-10 days after feeding of trophozoites; and in 8 out of 17 cats, 20-24 days after feeding of cat feces containing oocysts. Oocysts and infectivity were present in similar numbers, and they disappeared simultaneously from the feces of cats. Oocysts and infectivity were also observed simultaneously in the feces of 9 kittens, 1-2 days old, fed Toxoplasma cysts. Oocysts could not be separated from infectivity by filtration, by continuous particle electrophoresis, or by density gradient centrifugation. Excystation of oocysts was followed by an increase in titer of Toxoplasma infectivity. Unsporulated oocysts in fresh cat feces were noninfectious to mice, but oocyst sporulation was associated quantitatively with the development of infectivity at different temperatures and conditions of oxygenation. Maximum oocyst sporulation at 48 hr correlated with the development of maximum Toxoplasma infectivity. 1 and 2% sulfuric acid, and 2.5% potassium dichromate were found to be the best preservatives for sporulation of oocysts and for the development of Toxoplasma infectivity. Low sporulation rates in 0.1% formalin, 20% ethanol, and in water were associated with low infectivity in these reagents. Neither Toxoplasma infectivity nor oocysts developed in 0.3% formalin, 1% ammonium hydroxide, or 1% iodine in 20% ethanol. Oocysts, sporocysts, and sporozoites were stained specifically with Toxoplasma antibody in the indirect fluorescent antibody test. Typical coccidian stages, schizonts, and male and female gametocytes were found in the epithelium of the small intestine of kittens fed Toxoplasma cysts. The classification of T. gondii is discussed in relation to that of other isosporan coccidia of cats and dogs. The term "Toxoplasma oocyst" is introduced and Toxoplasma is classified in the family Toxoplasmidae of the suborder Eimeriina. The species Isospora bigemina is restricted to dogs, and I. cati to cats. I. felis and so-called I. rivolta from cats were noninfectious to dogs, and did not confer immunity to subsequent infection with I. canis and I. rivolta from dogs.

Air↗
PubMed1996

Viability and infectivity of Cryptosporidium parvum oocysts are retained upon intestinal passage through a refractory avian host.

Six Cryptosporidium-free Peking ducks (Anas platyrhynchos) were each orally inoculated with 2.0 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. Histological examination of the stomachs jejunums, ilea, ceca, cloacae, larynges, tracheae, and lungs of the ducks euthanized on day 7 postinoculation (p.i.) revealed no life-cycle stages of C. parvum. However, inoculum-derived oocysts extracted from duck feces established severe infection in eight neonatal BALB/c mice (inoculum dose, 2.5 x 10(5) per mouse). On the basis of acid-fast stained direct wet smears, 73% of the oocysts in duck feces were intact (27% were oocyst shells), and their morphological features conformed to those of viable and infectious oocysts of the original inoculum. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the feces-recovered oocysts (the majority were 3+ to 4+). The dynamics of oocyst shedding showed that the birds released a significantly higher number of intact oocysts than the oocyst shells (P < 0.01). The number of intact oocysts shed (87%) during the first 2 days p.i. was significantly higher than the number shed during the remaining 5 days p.i. (P < 0.01) and significantly decreased from day 1 to day 2 p.i. (P < 0.01). The number of oocyst shells shed during 7 days p.i. did not vary significantly (P > 0.05). The retention of infectivity of C. parvum oocysts after intestinal passage through an aquatic bird has serious epidemiological and epizootiological implications. Waterfowl may serve as mechanical vectors for the waterborne oocysts and may enhance contamination of surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to watershed management practices, the watershed protection program should consider waterfowl as a potential factor enhancing contamination of the source water with C. parvum.

Animals↗
PubMed1999

Method for detection and enumeration of Cryptosporidium parvum oocysts in feces, manures, and soils.

Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 10(2), 10(3), 10(4), and 10(5) oocysts g of bovine feces-1. The percentages of recovery ranged from 10.8% (25 oocysts g-1) to 17.0% (10(4) oocysts g-1) (r2 = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces-1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (10(4) oocysts g-1). The percentages of recovery were highest for bovine feces (17. 0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris-0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil-1 depending on the soil type. The percentages of recovery without dispersant (distilled H2O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.

Animals↗
PubMed2000

An immunoglobulin G1 monoclonal antibody highly specific to the wall of Cryptosporidium oocysts.

The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG(1) monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.

Animals↗