Abstract:
Cryptosporidium parvum is an apicomplexan protozoan parasite which causes diarrhea in both human and wide range of animals. Since this protozoa causes remarkable economic losses in cattke industry in Iran, the molecular determination of porotozoa and characterization of its protein pattern and immunogenic antigend are the aim of this study. In this study, diarheatic fecal samples of calves suspected for cryptosporidiosis were collected and identified. Purification and concentration of cryptosporidial oocysts from fecal samples was performed. Oocysts were confirmed as Cryptosporidium parvum by semi-nested PCR using specific primers designed from 18srRNA gene of Cryptosporidium parvum. A calf with negative antibodies against Cryptosporidium parvunm was infected with 5 ×106 oocysts. 5 days after inoculation, oocysts were isolated and purified . Soluble proteins from sporozoites were prepared and analyzed by SDS-PAGE and western blotting. There was an intense recognition of some10 to 100 kDa, ten low molecular weight proteins were recognized between 20-40 kDa, six separated protein bands was recognized between 40-70 kDa, immunoreactive proteins were present at different molecular weights between 17-260 kDa. Three antigens of apparent molecular weights 20-30 kDa, three antigen bands between 40-60 kDa and 2 bands 70-75 kDa were identified. Antibody responses to cryptosporidial antigens at high molecular weights were successfully diagnosed with apparent molecular weights 130,170,216 and 257kDa.
Key words: Cryptosporidium parvum, Immunoblot, SDS-PAGE, PCR, Semi-nested PCR.