[Identification of calf-origin Cryptosporidium bovis Shanghai isolate by nested PCR].

Objective: To identify a strain of Cryptosporidium in the feces of naturally infected calf in Shanghai.

Methods: Stool sample was examined by modified acid-fast staining. The size and morphology of the oocysts were microscopically determined. Genomic DNA was extracted from the oocysts isolated from feces of a naturally Cryptosporidium-infected calf. According to the sequence of Cryptosporidium 18S rRNA gene, two pairs of primers were designed and synthesized. The PCR products was amplified by nested PCR and sequenced in double directions. Homology searches were done over the Web using the program Blast. Phylogenetic tree was constructed with NJ method by MEGA4.0 software.

Results: Oocysts of the Shanghai isolate were round or elliptical with a size of (5.6 +/- 0.49) microm x (5.2 +/- 0.51) microm. Nested PCR resulted in fragments of approximately 810 bp, and the 18S rRNA nucleotide sequence had 100% identity with C. bovis from Brazil (GenBank Accession No: 151935628). This isolate was clustered in the same clade with C. bovis from Brazil. It showed an identity of 99% with the sequences of C. bovis from Qinghai Province of China, Mongolia, USA, and Tunisia.

Conclusion: The calf-origin Cryptosporidium derived from Shanghai has been identified as C. bovis.