*4.2. C. felis Isolates*

The nested PCR targeting the small subunit (SSU) rRNA gene was used to detect *Cryptosporidium* spp. [29]. DNA preparations of 30 *C. felis*-positive fecal samples from China were used in the present study. Among them, 18 samples were obtained from cats in Guangdong province, including 7 from pet shelters, 6 from animal hospitals, 4 from stray cats, and 1 from a pet shop. The remaining 12 samples were obtained from cats in Shanghai, including 8 from pet shelters and 4 from pet shops. They were from two previous studies of molecular epidemiology of cryptosporidiosis in cats [27,28].

#### *4.3. PCR Amplification*

The newly developed nested PCR targeting the conserved region of the *gp60* gene was employed to identify the subtypes of *C. felis* in this study [17]. Briefly, primers GP60- Felis-F1 (5 -TTT CCG TTA TTG TTG CAG TTG CA-3 ) and GP60-Felis-R1 (5 -ATC GGA ATC CCA CCA TCG AAC-3 ) were used in primary PCR, while GP60-Felis-F2 (5 -GGG CGT TCT GAA GGA TGT AA-3 ) and GP60-Felis-R2 (5 -CGG TGG TCT CCT CAG TCT TC-3 ) were used in secondary PCR. The sizes of primary and secondary PCR products were approximately 1200 and 900 bp, respectively. The PCR reaction and cycling program were described recently [18]. Each DNA preparation was analyzed in duplicate, with the inclusion of both positive (*C. felis* DNA) and negative (reagent-grade water) controls in each PCR run. The positive products from the secondary PCR were identified by 1.5% agarose electrophoresis.

#### *4.4. DNA Sequence Analyses*

All positive products of the expected size were sequenced bidirectionally on an ABI3730 autosequencer by the Sangon Biotech (Shanghai, China) using secondary PCR primers. The DNA sequences generated were assembled using ChromasPro 2.1.6 (http://technelysium. com.au/wp/) and edited using BioEdit 7.1.3.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit. html). These and the reference sequences from GenBank were aligned with each other using the MUSCLE program implemented in MEGA 6 (https://www.megasoftware.net/). Tandem Repeats Finder 4.09 (http://tandem.bu.edu/trf/trf.html) was used to identify repetitive sequences within them. A maximum-likelihood tree was constructed using MEGA 6 based on substitution rates calculated using the general time reversible model and gamma distribution. The bootstrap method with 1000 replicates was used to assess the reliability of the phylogenetic clusters formed. Representative nucleotide sequences of the *C. felis* subtypes identified in the present study were deposited in the GenBank database under accession numbers MW351820-MW351832.

#### **5. Conclusions**

The present study reported the subtype characteristics of *C. felis* isolates from cats in China for the first time. The results of the phylogenetic analysis suggested the potential zoonotic transmission of this pathogen. More isolates from diverse areas and hosts should be analyzed to confirm this conclusion.

**Author Contributions:** Conceptualization, L.X. and Y.F.; data curation, J.L.; formal analysis, J.L.; funding acquisition, L.X. and Y.F.; investigation, J.L., F.Y., R.L. and S.G.; methodology, L.X. and Y.F.; project administration, N.L.; resources, Y.F.; software, L.X. and Y.F.; supervision, Y.G.; validation, L.X. and Y.F.; visualization, L.X. and Y.F.; writing—original draft, J.L.; writing—review and editing, L.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Guangdong Major Project of Basic and Applied Basic Research (2020B0301030007), by the National Natural Science Foundation of China (U1901208 and 31820103014), by the 111 Project (D20008), and by the Innovation Team Project of Guangdong University (2019KCXTD001).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.
