Cryptosporidium, Tyzzer, 1907

Kubota, Rie, Tokiwa, Toshihiro, Matsubara, Katsuki, Okamoto, Minoru & Ike, Kazunori, 2020, Detection and molecular characterization of Cryptosporidium species in wildcaught pet spiny-tailed lizards, International Journal for Parasitology: Parasites and Wildlife 11, pp. 83-87 : 84

2.3. Molecular analyses of the Cryptosporidium

Fecal specimens from both lizards were placed in separate vials containing 2.5% aqueous potassium dichromate (K 2 Cr 2 O 7) solution and were submitted to molecular analyses at Nippon Veterinary and Life Science University, Tokyo, Japan.

Total genomic DNA was extracted using PowerSoil DNA Isolation Kit (Mo Bio Laboratories, USA) according to the manufacturer's instructions and used as a template for PCR analysis. For genotyping of the Cryptosporidium species, a nested PCR assay that targeted the partial fragment of the nuclear small subunit ribosomal RNA (SSU) gene was amplified as previously described by Sevá Ada et al. (2011). The nested PCRs targeting the partial fragment of actin and 70-kDa heat shock protein (HSP70) genes were amplified and sequenced using specific primers for subtyping analysis ( Sulaiman et al., 2000, 2002). Primary and secondary PCR reactions were carried out in a volume of 25 μl and 50 μl respectively, containing 10 × Ex Taq buffer, 2.5 mM of each dNTP mixture, 400 nM of forward and reverse primers, 5 units of TaKaRa ExTaq (TaKaRa Shuzo Co. Ltd., Otsu, Japan), and 1 μl of DNA template or primary PCR product. Amplification of the SSU gene involved the following, the templates were subjected to an initial denaturation at 94 ̊C for 3 min followed by 35 cycles of 94 ̊C for 45 s, 60 ̊C for 45 s (for the primary amplification) or 56 ̊C for 90 s (for the second amplification), and 72 ̊C for 60 s, a final extension at 72 ̊C for 5 min. Furthermore, to determine the primary reaction of actin and the HSP70 genes, the reaction conditions were an initial denaturation at 94 ̊C for 5 min followed by 35 cycles of 94 ̊C for 45 s, 50 ̊C for 45 s, and 72 ̊C for 60 s, a final extension at 72 ̊C for 10 min. The second amplification was altered slightly and the annealing temperature was 45 ̊C for actin and 55 ̊C for HSP70. The PCR products from the second amplification were analyzed using 1.5% agarose gel electrophoresis and sequenced with Applied Biosystems 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) at Macrogen Japan (Kyoto, Japan) using the secondary primers.

Sequence similarity was determined using BLAST analysis from the National Center for Biotechnology Information website. In order to construct a phylogenetic tree, the present sequences were aligned with reference sequences obtained from DDBJ/ENA/GenBank using MAFFT version 7 online service with the option Q-INS-I setting ( Katoh and Standley, 2013), followed by a manual edit. The concatenated dataset of SSU, actin, and HSP70 genes sequences were then aligned with the reference ( Holubová et al., 2016). Selection of the optimum DNA/ Protein models using the MEGA version 7.0 software was the first step of phylogenetic analyses ( Kumar et al., 2016). Then the phylogenetic tree was inferred by neighbor-joining (NJ) and the maximum likelihood (ML) methods. All positions contained gaps and the missing data were eliminated. Bootstrap support for branching was based on 1000 replicates.

The partial nucleotide sequence of the SSU, actin, and HSP70 gene sequences for Cryptosporidium sp. detected from the Arabian blue mastigure have been deposited in the DNA Data Bank of Japan (DDBJ) under the accession numbers, LC416466 (401bp), LC416467 (1,831bp) and LC416468 (918bp).