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This project constitutes a study to compare diagnostic procedures (faecal examination and biopsies) for the detection of Cryptosporidium infection in the host. The infectivity of oocysts of Cryptosporidium parvum was examined. Oocysts, obtained from faecal samples of infected cattles, were purified using sucrose gradients. Oocysts in the study were four to six weeks old when they were used for the UV disinfection and infectivity experiments. The oocysts were suspended in deionized water and exposed to well-defined doses of ultraviolet (UV) light (254 mm). The experiments were carried out under laboratory conditions in a specially constructed apparatus. Oocyst concentrations of 1 x 106 per ml in a total volume of 30 ml in Petri dishes were used and four selected UV dosages (300, 150, 75, 30 J/m2) were applied. The oocyst survival rate was measured after in vivo infectivity assay using the SCID mice model and compared to the infectivity rate by unexposed controls. SCID mice (C.B-17-SCID/IcrCrl, male and female, 4 weeks old) were supplied by Charles River GmbH, Germany. Several groups were used with usually five animals in each group. Each mouse of the group was orally administered an aliquot of 0,5 ml of the appropriate inoculum between 2,5 . 10 x 105 (250,000, 450,000 and 500,000, 1,000,000) oocysts from the exposed sample as have been previously described. Each inoculated animal was kept in a separate cage. Two diagnostic techniques have been applied for the detection of Cryptosporidium oocysts: faecal samples from inoculated mice were collected between 5 to 18 days post inoculation (dpi), diluted in distilled water, and oocysts were separated using discontinuous sucrose gradient and were then detected by Direct Immunofluorescence (DIF), (Crypto-CELL, Cellabs Pty.); and, mice were sacrificed 15-18 days after inoculation by cervical dislocation. Intestinal manifestations and histopathology of the small intestine in comparison to the uninfected animals and to the results from the faecal examinations are also described. Pieces of the small intestine (duodenum, jejunum, and ileum) of each mouse from each experiment have been prepared for histopathology analysis. Tissues were fixed in formaldehyde, embedded in paraffin and thins sections (3-5 um) stained with HE-method. Hematoxylin was used for nuclear-staining (10-12 min) and azophloxin for cytoplasma-staining (7 min). The sections are collered in violet-red. Cryptosporidium oocysts are stained violet-blue. The sections were observed by x100 and x400 magnification. Includes 10 references, tables.

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Edition: Vol. - No. Published: 09/22/2002 Number of Pages: 9File Size: 1 file , 150 KB