*2.2. Identification of the gp60 Gene of C. xiaoi*

To obtain the nucleotide sequence of the *gp60* gene of *C. xiaoi*, we conducted wholegenome sequencing of one isolate (SCAU2942) from a Hu sheep in Anhui, China using the established procedures [21]. The genome was sequenced using Illumina HiSeq 2500 analysis of an Illumina TruSeq (v3) library with 250-bp paired-end reads. The sequence reads were assembled de novo using the SPAdes version 3.13 (http://cab.spbu.ru/software/ spades/, accessed on 21 November 2019) with a K-mer size of 63. The *gp60* gene of *C. xiaoi* was identified by the blastn analysis of the genome assembly with the *gp60* (cgd6\_1080) sequence of *C. parvum*. The coding region and amino acid sequence of the *gp60* gene were predicted using the combination of FGENESH (http://www.softberry.com/berry.phtml, accessed on 15 December 2019) and blastp search of the NCBI database.

#### *2.3. Subtyping of C. xiaoi*

Based on the sequence of the *C. xiaoi gp60* gene, nested PCR primers were designed for the subtyping analysis. The primers used in primary and secondary PCR were Xiaoi-*gp60*- F1 (5 -CCTCTCGGCACTTATTGCCCT-3 ) and Xiaoi-*gp60*-R1 (5 -ATACCTGAGATCAAAT GCTGATGAA-3 ), and Xiaoi-*gp60*-F2 (5 -CCTCTTAGGGGTTCATTGTCTA-3 ) and Xiaoi*gp60*-R2 (5 -TACCTTCAAAGATGACATCAC-3 ), respectively. Each PCR was performed in a 50 μL-reaction containing 1×PCR master mix (Thermo Scientific, Waltham, MA, USA), 0.25 μM primary PCR primers or 0.5 μM secondary PCR primers, and 1 μL of DNA (primary PCR) or 2 μL of the primary PCR product (secondary PCR). To reduce PCR inhibitors, 400 ng/μL of nonacetylated bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) was used in the primary PCR. The PCR amplification consisted of an initial denaturation at 94 ◦C for 5 min; 35 cycles of 94 ◦C (denaturation) for 45 s, 55 ◦C (annealing) for 45 s, and 72 ◦C (extension) for 90 s; and a final extension of 72 ◦C for 10 min. The secondary PCR products were visualized by 1.5% agarose gel electrophoresis.

*Pathogens* **2021**, *10*, 800


Samples from a previous study [19].

#### *2.4. DNA Sequence Analysis*

All secondary *gp60* PCR products were sequenced in both directions using Sanger sequencing by Sangon Biotech (Shanghai, China). For the samples yielding double PCR bands with different sizes, PCR products of each band were excised from the agarose electrophoresis gel and purified using the E.Z.N.A.® Gel Extraction Kit (Omega bio-tek, Norcross, GA, USA) before sequencing. The sequences obtained were assembled using ChromasPro 1.5 (http://technelysium.com.au/wp/chromaspro/, accessed on 20 March 2020), edited using BioEdit 7.1 (http://www. mbio.ncsu.edu/bioedit/bioedit, accessed on 20 March 2020), and aligned with reference sequences from GenBank using MUSCLE in MEGA 7.0 (https://www.megasoftware.net/, accessed on 20 March 2020). Short tandem repeats in the gene were identified using the Tandem Repeat Finder (http://www.tandem.bu.edu/ trf/trf, accessed on 21 March 2020). The signal peptide and glycosylphosphatidylinositol (GPI) anchor were predicted using PSORT II (http://psort.hgc.jp/form2.html, accessed on 22 March 2020). N-glycosylated sites, O-glycosylated sites, and furin proteolytic cleavage sites were predicted using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/, accessed on 22 March 2020), YinOYang 1.2 (http://www.cbs.dtu.dk/services/YinOYang/, accessed on 22 March 2020), and ProP 1.0 (http://www.cbs.dtu.dk/services/ProP/, accessed on 22 March 2020), respectively. To assess the genetic relationship of *C. xiaoi* subtype families, a phylogenetic tree was conducted using the maximum likelihood (ML) analysis in MEGA 7.0 based on substitution rates calculated with the general time-reversible model. DnaSP 5.10 (www.ub.es/dnasp/, accessed on 25 March 2020) was used to calculate the recombination rates among subtype families of *C. xiaoi*.

#### *2.5. Nucleotide Sequence Accession Numbers*

Representative nucleotide sequences of the *C. xiaoi gp60* gene generated in this study were deposited in GenBank under accession numbers MW589389, MW815183-MW815276.
