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[Isolation and identification of an isolate of cow-origin Cryptosporidium sp]
Hai-Peng Liu1, Jian-Ping Cao, Yu-Juan Shen
1National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Key Laboratory of Parasite and Vector Biology, MOH, Shanghai 200025, China.
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.
