Fluorescence detection of Cryptosporidium oocysts in human fecal specimens by using monoclonal antibodies.
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With the discovery that the coccidian parasite Cryptosporidium sp. can cause severe symptoms in humans, implementation of many diagnostic techniques rapidly followed. The infection is self-limiting in patients with normal immune systems but chronic in the immunosuppressed patient. With the eventual development and use of therapeutic agents, it will become very important to find Cryptosporidium sp., even in low numbers, in fecal specimens. Production of a highly specific and sensitive antibody by use of cloning techniques has provided another diagnostic tool. Formalinized positive human fecal specimens (n = 99) and negative specimens (n = 198), of which 115 contained yeastlike fungi and other organisms, were tested in blind trials by use of a monoclonal antibody. Sensitivity was 100% with 3- to 4+ fluorescence on all cryptosporidial oocysts, both in light and heavy infections. The organisms were round and easily visible (4 to 6 micron), showing apple-green to yellow fluorescence against a dark background free of nonspecific fluorescence. Specificity was also 100% with all 99 positive Cryptosporidium sp. specimens exhibiting fluorescence and all 198 negative specimens showing no fluorescence. All positive and negative specimens were previously confirmed by the hot modified acid-fast technique. However, seven specimens previously considered negative by this acid-fast method were positive by the monoclonal antibody technique. These specimens were confirmed as positive, after extensive examination of additional smears prepared by the modified hot acid-fast method revealed rare organisms, emphasizing the increased sensitivity of the monoclonal antibody technique. Since acid-fast stains do not always consistently stain all oocysts, the increased sensitivity of the monoclonal reagent provides an excellent screening method. (+info)
Identification of Cryptosporidium oocysts in river water.
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Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission. (+info)
Detection of Cryptosporidium in water by using polypropylene cartridge filters.
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Members of the genus Cryptosporidium are protozoan parasites that cause gastroenteritis in humans and animals and appear to be spread largely by the fecal-oral route. A method was developed for the concentration and detection of Cryptosporidium oocysts in water to assess their occurrence in the environment and potential for waterborne disease transmission. This method was developed by using spun polypropylene cartridge filters. Optimal conditions for concentration, filter elution, filter porosity, and detection were determined. Fluoresceinated monoclonal antibodies were used for oocyst detection. Experiments also were conducted to study the effect of flow rate, low oocyst numbers, and the addition of detergents on recovery and retention of oocysts. The method that was developed was sensitive enough to detect oocysts at levels of less than 1 per liter. Using this method, we isolated Cryptosporidium oocysts from secondarily treated sewage. (+info)
Abomasal cryptosporidiosis in cattle.
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A 6-week-old calf and nine feedlot steers shed oocysts similar to Cryptosporidium muris-like oocysts. There were massive populations of this Cryptosporidium in the peptic glands of most of these animals. The oocysts were larger and more oval than the frequently reported type which is generated in the intestines of many animal species and thought to be similar to Cryptosporidium parvum. The pattern of shedding of this newly discovered Cryptosporidium in the steers was continuous over a period of months whereas the C. parvum-like oocysts cease to be shed 1 to several weeks after onset. The nature of the host-parasite interaction in abomasal cryptosporidiosis is yet to be determined. Morphologic changes that resulted from the interaction were an approximate 10% increase in abomasal mucosal thickness, widening of gland lumens in the middle region, and atrophy of epithelium in the same region. (+info)
Pathogenesis of neurological signs associated with bovine enteric coccidiosis: a prospective study and review.
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Various hypotheses have been proposed for the pathogenesis of the neurological signs associated with bovine enteric coccidiosis. We undertook a prospective study of cases of bovine enteric coccidiosis with and without nervous signs to test the validity of these hypotheses and explore other possible pathophysiological mechanisms. Clinical, pathological and toxicological data from 12 calves with, and 15 calves without, neurological signs were compared. Calves with neurological signs had a lower liver Cu concentration (p less than 0.01) and a higher plasma glucose concentration (p less than 0.05) than did calves without neurological signs. Hyperglycemia and Cu deficiency may increase the susceptibility to central nervous system damage, but are not likely to account for the onset of neurological signs in calves with enteric coccidiosis. The results of the study suggest that the following are not involved in the pathogenesis of "nervous coccidiosis": disturbance of serum Na, K, Ca, P, or Mg concentration, vitamin A deficiency, thiamine deficiency, anemia, lead intoxication, uremia, Haemophilus somnus meningoencephalitis, severity of coccidial infection, gross alterations in intestinal bacterial flora and hepatopathy. (+info)
Separation of Cryptosporidium species oocysts from feces by using a percoll discontinuous density gradient.
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Cryptosporidium oocysts were concentrated by an ether-phosphate-buffered saline sedimentation technique and then separated by density gradient centrifugation. This two-step method yielded highly concentrated oocysts largely free of bacteria and fecal debris. (+info)
Separation of cryptosporidium oocysts from fecal debris by density gradient centrifugation and glass bead columns.
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A method was developed to obtain purified cryptosporidium oocysts from fecal samples. Oocysts were initially collected by centrifugation through a sucrose density gradient and further purified by passage through glass bead columns. The purified oocysts were antigenically active and sufficiently pure for immunological studies. (+info)
Experimental intrauterine infection of adult BALB/c mice with Cryptosporidium sp.
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Inoculation of adult, female BALB/c mice with 2 X 10(5) bleach-treated Cryptosporidium sp. oocysts isolated from calf feces resulted in infection of the uterine mucosa in more than 50% of the animals. Cryptosporidium sp. completed the entire life cycle in the uterus, and infectious oocysts were passed into the vagina. Two methods of application were used to establish intrauterine infection. The inoculum was either injected into the uterus after abdominal surgery or intracervically instilled. Mice were susceptible at all phases of the sexual cycle, but the highest infection rates were obtained during estrus and diestrus. Parasites were demonstrated as early as 5 days postinfection. Phagocytic cells in the uterine lumen and in the vagina contained Cryptosporidium sp. Phagocytosis may be an important immune response and a mechanism of parasitic clearance. These results suggest that Cryptosporidium sp. is a potential pathogen of the reproductive tract. (+info)