Cryptosporidium

Xiao, Xiao, Qi, Rui, Han, Hui-Ju, Liu, Jian-Wei, Qin, Xiang-Rong, Fang, Li-Zhu, Zhou, Chuan-Min, Gong, Xiao-Qing, Lei, Si-Cong & Yu, Xue-Jie, 2019, Molecular identification and phylogenetic analysis of Cryptosporidium, Hepatozoon and Spirometra in snakes from central China, International Journal for Parasitology: Parasites and Wildlife 10, pp. 274-280 : 275

2.1. Microscopy screening and concentration of Cryptosporidium

Fecal or large intestinal content samples were mixed with phosphate-buffered saline (PBS) to make homogeneous suspensions. The suspensions were sequentially sieved and finally purified through 63- μm porosity mesh stainless steel screens. After concentration using Sheather's discontinuous sucrose gradient centrifugation technique as previously described ( Arrowood, 2020), all supernatants were examined by optical microscopy under 250× magnification for the screening of Cryptosporidium oocysts based on the morphological standard previously reported ( Arrowood and Sterling, 1987).

2.2. DNA extraction

The supernatants containing Cryptosporidium oocysts were repeatedly frozen and thawed in liquid nitrogen and a 65 ̊C water bath, respectively, before centrifugation ( Wang et al., 2014b). The total DNA was extracted using the QIAampṜ DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. For blood samples, genomic DNA was extracted using a Qiagen DNeasy Blood & Tissue Kits (Qiagen, Germany) according to the manufacturer's instructions. Total genomic DNA of sparganas was extracted from representative sparganas isolated from different infected snake species using a DNeasy Tissue Kit (Qiagen, Germany) according to the manufacturer's instructions. All purified DNA samples were stored at −20 ̊C for further molecular analysis.

2.3. PCR amplification and sequencing

Nested PCR was performed to identify Hepatozoon and Cryptosporidium species based on the 18S small subunit rRNA (SSU rRNA) gene sequence ( Aydin et al., 2015; Sumrandee et al., 2015; Xiao et al., 2004). DNA from Cryptosporidium SSU rRNA-positive samples was subjected to further PCR-based analysis targeting the 60-kDa glycoprotein (gp60) gene ( Alves et al., 2003). The highly conserved cox1 gene was targeted for identification of Spirometra species using conventional PCR ( Lee et al., 2007). The primer sequences, product lengths, and the annealing temperatures are listed in Table 1. The previously isolated DNA of Spirometra erinaceieuropaei and Cryptosporidium parvum were used as positive controls, while reagent-grade water served as a negative control in each run. All steps were performed in separate rooms to avoid contamination. The secondary PCR products were examined by electrophoresis using a 1.2% agarose gel and staining with ethidium bromide, and observed under UV light. PCR products with expected sizes were excised from gels and extracted using a Gel Extraction Kit (Promega, Madison, WI, USA). The PCR products were cloned into the pMD 19-T vectors (TaKaRa, Shiga, Japan) and recombinant clones were sequenced bidirectionally with Sanger sequencing.