*4.3. DNA Extraction and PCR Amplification*

The fecal specimens were washed twice with distilled water by centrifugation to remove ethanol before DNA extraction. Extraction of genomic DNA from specimens was performed using the FastDNA SPIN Kit for Soil (BIO 101, Carlsbad, CA, USA). The genomic DNA was eluted with 100 μL reagent-grade water and stored at −20 ◦C until PCR analysis.

#### *4.4. Cryptosporidium Detection, Genotyping and Subtyping*

*Cryptosporidium* spp. in the specimens were detected by nested PCR analysis of a ∼830-bp fragment of the small subunit rRNA (*SSU rRNA*) gene as previously described [49]. *Cryptosporidium* species were identified by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products of *SSU rRNA* gene using *Ssp*I (New England BioLabs, Ipswich, MA, USA) and *Vsp*I (Promega, Madison, WI, USA) restriction enzymes [50]. All *Cryptosporidium*-positive specimens were selected for further subtyping by PCR and sequence analysis of the 60-kDa glycoprotein (*gp60*) gene [51]. Each specimen was analyzed twice for each genetic target, using *C. baileyi* DNA as the positive control for the *SSU rRNA*-based PCR, *C. parvum* DNA as the positive control for *gp60*-based PCR, and reagent-grade water as the negative control for both PCR assays.

#### *4.5. DNA Sequence and Phylogenetic Analysis*

Montage PCR filters (Millipore, Bedford, MA, USA) were used to purify all secondary PCR products of both genes. The purified products were sequenced in both directions using the secondary PCR primers and Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130 Genetic Analyzer (Applied Biosystems). ChromasPro (version 1.5) (www.technelysium.com.au/ChromasPro.html/, accessed on 22 June 2009) was used to edit and assemble the DNA sequences, while ClustalX 2.0.11 (http://www.clustal.org/, accessed on 1 June 2018) was used to align the obtained nucleotide sequences against each other and reference sequences from GenBank to determine the genetic relatedness of various *C. cuniculus* subtype families. A phylogenetic tree was constructed using the maximum likelihood algorithm implemented in MEGA version 7.0.26 (www.megasoftware.net/, accessed on 1 May 2017) based on substitution

rates calculated with the general time reversible model. Bootstrap analysis was applied to evaluate the reliability of cluster formation in the phylogenetic tree with 1000 replicates.

#### *4.6. Statistical Analysis*

Differences in infection rates of *Cryptosporidium* spp. among rabbits of different age groups, localities, and breeds were estimated using the Fisher's exact test. The SPSS software version 20.0 (IBM, Armonk, NY, USA) was used in the statistical analysis of the data. Differences were considered significant at *p* ≤ 0.05.

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

**Funding:** This study was supported partially by the National Natural Science Foundation of China (31820103014), 111 Project (D20008), Innovation Team Project of Guangdong University (2019KCXTD001), and the Centers for Disease Control and Prevention.

**Institutional Review Board Statement:** Permission was obtained from the owners of the farms before collections of fecal specimens. All fieldwork associated with this study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals in Egypt. The study protocol was approved by the Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, Egypt.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All relevant data are within the paper.

**Acknowledgments:** The findings and conclusions in this report are those of the authors and do not necessarily represent the represent of the official position of the U.S. Centers for Disease Control and Prevention.

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

#### **References**


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