**2. Results**

#### *2.1. Amplification of the gp60 Gene*

Among the 30 DNA preparations that were positive for *C. felis* based on nested PCR analysis of the small subunit (SSU) rRNA gene, 20 (66.7%) generated the expected products in the *gp60* PCR. Among them, 9 were from cats in pet shelters, 5 were from cats visiting animal hospitals, 4 were from cats in pet shops, and 2 were from stray cats (Table 1). These PCR products differed slightly in size (Figure 1) and were all sequenced successfully.


**Table 1.** Sources of *Cryptosporidium felis* samples used in the study and their *gp60* subtype identity.

N/A: PCR negative.

#### *2.2. Nucleotide Sequence Variations in the gp60 Gene of C. felis*

A comparison of the nucleotide sequences generated led to the identification of 15 sequence types. They differed from each other by both nucleotide substitutions and copy numbers of repetitive sequences. The sequence alignments showed the presence of numerous single nucleotide substitutions (SNPs) over the partial *gp60* gene among the 20 *C. felis* isolates. Three types of simple tandem repeats were detected in the *gp60* genes (Table 2). Among them, 2–5 copies of the 33-bp repeat sequence 5 -CCACCTAGTGGCGGTAGTGGCGTGTCCCCTGCT-3 and 2– 4 copies of the 39-bp repeat sequence 5 -CCACCTAGTGGCGGTAGTGGCGTGTCCCCTGCT-3

were observed at nucleotides 460–577 and 778–911 of the sequence alignment, respectively. In both repeat types, the last copy had only half of the usual length. In addition, 5 copies of the trinucleotide repeat GTT were found in the *gp60* sequences at nucleotides 1143-1154.

**Figure 1.** Analysis of the 60-kDa glycoprotein (*gp60*) gene in *Cryptosporidium felis* by nested PCR: lanes 1 and 2, SCAU320; lanes 3 and 4, SCAU2396; lanes 5 and 6, SCAU1149; lanes 7 and 8, SCAU1850; lanes 9 and 10, SCAU1851; lanes 11 and 12, SCAU1857; lane M, 100-bp molecular marker; P1 and P2, positive control (*C. felis* DNA); and N1 and N2, negative control (reagent-grade water).


**Table 2.** Tandem repeats in nucleotide sequences of the *gp60* gene of *Cryptosporidium felis.*

<sup>a</sup> Sample IDs labelled with ECUST were from cats in Shanghai, while those with SCAU were from cats in Guangdong; <sup>b</sup> 33-bp tandem repeat (5 -CCACCTAGTGGCGGTAGTGGCGTGTCCCCTGCT-3 ) with a partial copy at the end; <sup>c</sup> 39-bp tandem repeat (5 -AGCACAACTGCGGCTACAGCGAGCACTGCGAGTTCGACA-3 ) with a partial copy at the end and 0–2 nucleotide differences.
