جداسازي و تخليص اووسيست ها و اسپروزوييت هاي كريپتوسپوريديوم با استفاده از روشهاي گراديانت منقطع ساكارز و گراديانت پركول

Isolation of cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic percoll gradients

Cryptosporidium spp. is an obligate intracellular protozoan parasite (protozoa, apicomplexa) that has been recognised worldwide as a major cause of gastrointestinal disease in a variety of mammalian hosts, including human. Cryptosporidium primerily infects epithelial cells of the gastrointestinal tract, resulting a variety of problems ranging from acute, self-limiting diarrhea to fatal cases in immunocompromised persons, particularly those with aquired immunodificiency (AIDS). Regarding to the public health importants of this parasite, isolation and purification of Cryptosporidium spp. is essential for the detection of antigenic characterization and characteristic of various strains. In the present study, a total 480 samples from calves were collected in Isfahan provience. 30 of these samples were infected with Cryptosporidium by using the Scheatherʹs flotation and Modified Ziel-Nelson Acid Fast methods. Fecal samples containing Cryptosporidium were purified using sucrose gradient centrifugation and were stored in 2.5% potassium dichromate solution at 4 °C. Techniques for the large-scale isolation of cryptosporidium oocysts and sporozoites, obtained from the feces of infected calves, were developed employing discontinuous sucrose gradients and isopycnic percoll gradients. The oocyst recovery method utilized 2 sequential discontinuous sucrose gradients followed by 1 percoll gradient. Recover oocysts were essentially free of debris and bacteria and represented 34% of the orginal oocyst suspension. Sporozoites were recovered from excystation mixtures on single percoll gradient. Sixthy-three peacid analysis.