Cryptosporidium spp

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 : 85

3.2. Sequence analysis of Cryptosporidium spp . from reptiles

The partial fragment of the SSU gene (401 bp) was successfully amplified and sequenced in both of the lizards. SSU sequences from two lizards were identical to each other and showed 100% identity (401/ 401 bp) with the Cryptosporidium sp. (accession no. LC310795) from a brown wood owl ( Strix leptogrammica ) in Japan ( Makino et al., 2018). Identities of 99.8% (400/401 bp, derived from a single adenine insertion) for C. avium (accession nos. KU058875 and JX548299) and avian genotype II (accession nos. KU058875 and KJ487974) were also noted. Phylogenetic analyses using SSU sequences showed that the Cryptosporidium from the mastigure and brown wood owl and C. avium were monophyletic and placed within a clade of avian-associated Cryptosporidium clade ( Fig. 2 ).

Partial nucleotide sequences of the actin (1831 bp) and HSP70 (918 bp) genes were successfully amplified and sequenced from the Arabian blue mastigure. The sequence at the actin gene was 99% identity (906/ 918bp) with Cryptosporidium sp. from the brown wood owl (accession no. LC310796) and C. avium from the common ostrich ( Struthio camelus ) (accession no. AB696815) and red-crowned parakeet ( Cyanoramphus novaezelandiae ) (accession no. KU058882). The sequence of the HSP70 gene showed 98.4% (1801/1831 bp) to 98.3% (1798/1829bp) identities with C. avium from several pet birds (accession nos. JQ798893, AB471665, MK311173). Moreover, an identity of 97.6% (1787/1831 bp) was found between this isolate and the brown wood owl (accession no. LC310797). The phylogenetic tree using concatenating data across multiple sequences of SSU, actin, and HSP70 genes revealed that the present species clustered with the Cryptosporidium species recorded from the brown wood owl, and this clade appeared to branch near the monophyletic C. avium clade ( Fig. 3 ).

Figures

Fig. 2. Phylogenetic relationships between Cryptosporidium sp. from the mastigures and other Cryptosporidium spp. The mid-point tree was generated with the neighbor-joining method using Tamura 3-parameter plus Gamma distribution. Nodal values represent boot strap values (> 50%) for neighbor-joining (left) and ML (right). Circle represents the major reptile-associated species. Bar represents the number of nucleotide substitutions per sites.

Fig. 3. Phylogenetic relationships of Cryptosporidium detected from the Arabian blue mastigure and closely-related species/subtypes as inferred by a maximum likelihood analysis of concatenated sequences were constructed from the partial DNA sequences of SSU (left), actin (middle), and HSP70 (right) loci. Numbers represent the boot strap values for NJ (left) and ML (Right). Bar represents the number of nucleotide substitutions per sites. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)