[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 primarily 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 immunodeficiency [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 province. 30 of these samples were infected with Cryptosporidium by using the Scheather's 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 oocytes 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 oocytes 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. Sixty-three peacid analysis

identification of Cryptosporidium species by analysis of PCR-RFLP of the 18s rRNA gene

Cryptosporidium is an important protozoa that cause. diarrheal illness in humans and animals. In immunocompetent individuals, infection is usually self-limiting; but in immunocompromised patients infections which can be life threatening may develop. Cryptosporidium may account for 10 to 20% of the cases of diarrhea in immunocompromised patients living in developed countries and as much as 50% in underprivileged countries. Different strains of Cryptosporidium have been reported, and it is believed that strain characteristics are an important factor to be considered in strategic planning for the control of cryptosporidiosis disease. In this study human and animal isolates of Cryptosporidium oocysts were examined by PCR-RFLP for identifying strain variation in Isfahan. A total of 642 fecal samples of children under 5 years of age, imunocompromised patients and high risk persons and 480 rectal specimens of cows and calves were selected randomly in Isfahan. After identification of the samples contaminated with the parasites, oocysts were purified from these samples, and their DNA were extracted. These DNAs were use for detection of Cryptosporidium species using PCR-RFLP analysis of a 1750bp region of 18srRNA. Microscopic results showed that 4.7% of human samples and 6.2% of animal samples were contaminated with Cryptosporidium. 18s rRNA gene of all isolates were amplified by PCR, and 18s rRNA fragment size of all isolates were identical approximately 1750 bp. RFLP results showed that the samples were contaminated with C. parvum II, C. baileyi, C. serpentis, C. muris, C. wrairi and three new genotypes of Cryptosporidium. PCR-RFLP results indicated the occurrence of most strains of Cryptosporidium specially C. parvum. The results also imply extensive polymorphism in these parasites and the occurrence of mutant strains of it. Furthermore, the occurrence of animal species of the parasite in human samples shows the importance of animal-human cycle of it