OBJECTIVE: To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. METHODS: Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheathed sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene (SSU rRNA) and Cryptosporidium oocyst wall protein gene (COWP), respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. RESULTS: The results suggested that the size of oocysts was (7.4+/-0.32) microm by (5.4+/-0.21) microm and the ratio of length and width was 1.37+/-0.07 (n=20). BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C. andersoni clade based on the sequence of SSU rRNA and COWP gene. CONCLUSION: What isolated from naturally infected cow feces has been identified as C. andersoni.
OBJECTIVE: To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. METHODS: Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheathed sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene (SSU rRNA) and Cryptosporidium oocyst wall protein gene (COWP), respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. RESULTS: The results suggested that the size of oocysts was (7.4+/-0.32) microm by (5.4+/-0.21) microm and the ratio of length and width was 1.37+/-0.07 (n=20). BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C. andersoni clade based on the sequence of SSU rRNA and COWP gene. CONCLUSION: What isolated from naturally infected cow feces has been identified as C. andersoni.