**3. Results**

#### *3.1. Features of the gp60 Gene of C. xiaoi*

A total of 25.85 million paired-end reads were obtained from the *C. xiaoi* isolate SCAU2942, and assembled into 334,080 contigs. The full *gp60* gene (MW589389) was identified in contig 1122 (8944 bp). The gene was 1437 bp in length and encoded 478 amino acids. Although it shared sequence similarities with the *gp60* gene of *C. parvum* (AF022929), *C. hominis* (FJ839883), *C. ubiquitum* [12], and *C. ryanae* [17] in the 5 and 3 regions at the amino acid level, the full sequence similarity was only 19.9 to 41.6% between *C. xiaoi* and the other four species (Figure 1). The GP60 protein of *C. xiaoi* had classic features of *Cryptosporidium* GP60 proteins, including an N-terminal signal peptide, a furin cleavage site (RSRR), two potential N-glycosylation sites, nearly 100 O-glycosylation sites in the GP40 region, and a GPI anchor at the C terminus. Nevertheless, the serine repeats (TCA/TCG/TCT) commonly seen in *C. parvum*, *C. hominis*, and related species, were absent in the 5 region of the *gp60* gene of *C. xiaoi*.

#### *3.2. Sequence Polymorphisms in the gp60 Gene of C. xiaoi*

Among the 434 samples positive for *C. xiaoi* in this study, the *gp60* gene in 355 samples (81.8%) was successfully amplified by PCR. PCR products of 323 samples generated one expected band in gel electrophoresis. However, 32 samples yielded two PCR bands with different sizes, including 16 sheep samples and 16 goat samples (Table 1 and Figure 2). All PCR products with either one or two bands were sequenced, generating 298 *gp60* nucleotide sequences with length ranging from 800 to 1170 bp. Nucleotide sequences from 18 samples were identical to the reference sequence (SCAU2942) from the whole-genome sequencing, while the remaining sequences were highly divergent and displayed nucleotide differences of 24.0–68.3% (Table 2). Altogether, 94 sequence types were identified among the 298 *gp60* sequences obtained. In addition to the numerous nucleotide substitutions observed over

the partial *gp60* gene, there was a significant length polymorphism among the 94 sequence types mostly due to the presence of repetitive sequences.


**Figure 1.** Deduced amino acid sequence of the *gp60* gene of *Cryptosporidium xiaoi* compared with sequences of *C. parvum* (AF022929), *C. hominis* (FJ839883), *C. ubiquitum* [12], and *C. ryanae* [17]. Potential *N*-glycosylation sites are indicated in bold and italic letters, and predicted O-glycosylation sites are indicated in bold and underlined letters. Amino acid sequences of the N-terminal signal peptide, furin cleavage site (RSRR), and C-terminal glycosylphosphatidylinositol anchor are highlighted in green, red, and blue, respectively. Dashes represent amino acid deletions.

**Table 2.** Pairwise nucleotide sequence similarity among subtype families of *Cryptosporidium xiaoi* in the *gp60* gene.


**Figure 2.** Nested PCR amplification of the partial *gp60* gene of *Cryptosporidium xiaoi* in sheep and goat samples. M: 100-bp DNA ladder. Lanes 1–20: Replicate PCR of 10 samples with divergent binding patterns. N-1 and N-2: No-template controls in the primary and secondary PCR, respectively.

#### *3.3. Subtype Families and Subtypes of C.xiaoi*

A total of 94 *gp60* sequences, including one sequence of each sequence type, were used in a phylogenetic analysis of the *gp60* gene. The ML tree generated comprised 12 clusters of sequences (Figure 3). They were named as subtype families XXIIIa–XXIIIl in concordance with the established nomenclature of *gp60* subtype families of *Cryptosporidium* spp. [22]. Subtype families XXIIIa–XXIIIh formed a group highly divergent from the other group of XXIIIi–XXIIIl (Figure 3). The nucleotide sequence differences among 12 subtype families ranged from 24.0 to 68.3% (Table 2). Among these subtype families, XXIIIl had the shortest nucleotide sequences and contained some unique AGC/AGT trinucleotide repeats encoding serine, leading to the occurrence of a long stretch of highly O-glycosylated amino acids. Subtypes within XXIIIl differed from each other mostly in the number of AGC/AGT trinucleotide repeats. The DnaSP analysis of the *gp60* nucleotide sequences revealed the presence of 71 potential recombination events among all 12 subtype families (Table 2). In addition, mosaic sequence patterns were observed among these subtype families (Figure 4).

At the amino acid level, extensive sequence polymorphism was found among 12 subtype families, mostly in the GP40 region (Figure 4). Despite the extensive sequence difference, all subtype families had the furin cleavage site of RSRR. There were one to four N-glycosylation sites in these subtype families, except for XXIIIk, which had none. In addition, the number of O-glycosylation sites was divergent among subtype families, with XXIIIb and XXIIIl having more O-glycosylation sites than other subtype families.

**Figure 3.** Phylogenetic relationship among 12 *Cryptosporidium xiaoi* subtype families (XXIIIa–XXIIIl) based on the maximum likelihood analysis of the partial *gp60* gene. General time-reversible model and Gamma distribution were used in the calculation of substitution rates. Bootstrap values lower than 50% are not displayed.


**Figure 4.** Deduced amino acid sequences of the partial *gp60* gene of 12 subtype families (XXIIIa–XXIIIl) in *Cryptosporidium xiaoi*. N-glycosylation sites are indicated in bold and italic letters, and O-glycosylation sites are indicated in bold and underlined letters. The furin cleavage site "RSRR" is highlighted in red. Dashes represent amino acid deletions (except those at both ends of the sequences).
