First report of Enterocytozoon bieneusi and Cryptosporidium spp. in peafowl (Pavo cristatus) in China

Feng, Sheng-Yong; Chang, Han; Luo, Jing; Huang, Jing-Jing; He, Hong-Xuan

The E.Z.N.A. ® Stool DNA Kit (Omega Biotek Inc., Norcross, USA) was used to extract genomic DNA from 200 mg fecal samples following the manufacturer's protocol. The extracted DNA was stored at −20°C until further PCR analysis. The small subunit ribosomal RNA (SSU rRNA) gene was amplified by nested PCR to identify Cryptosporidium species/genotypes as described by Nolan et al. (2010) . To detect E. bieneusi, a fragment of ITS was amplified via nested PCR in accordance with previous methods (Buckholt et al., 2002) . The obtained sequences were aligned with each other and reference sequences downloaded from the GenBank database with ClustalX 1.83 software package to differentiate E. bieneusi genotypes. All the primers used in this study were listed in Table 1 . Both negative (reagent-grade water) and positive controls (DNA extracted from the E. bieneusi PtEbIX genotype and C. baileyi) were included in each amplification to ensure the accuracy of the results. The secondary PCR products were detected by 2% agarose gel electrophoresis with GoldView™ (Solarbio, China) stained.