*3.5. Distribution of C. xiaoi Subtype Families by Farm*

One to 10 subtype families were found on each farm. As shown in Table 1, three farms had only one subtype family, two farms had two, two farms had four, one farm had six, one farm had seven, and two farms had 10. On Farms 9, 10, and 11 in Guangdong, all *gp60* sequences obtained belonged to the subtype family XXIIIa. In contrast, although only eight samples were subtyped on Farm 4 in Qinghai, they belonged to four subtype families (XXIIId, XXIIIe, XXIIIh, and XXIIIi). In addition, co-infections of different subtype families were observed in animals on Farms 1, 3, 5, 6, and 7, mostly with prevalent subtype families on the farm (Table 1).

#### **4. Discussion**

In the present study, we conducted whole genome sequencing of *C. xiaoi* and identified its *gp60* gene. Based on the sequence data, we established a *gp60*-based subtyping tool to assess the genetic diversity of *C. xiaoi*. The application of this subtyping tool in the analysis of *C. xiaoi*-positive samples from various breeds of sheep and goats has identified high genetic diversity within the species and possible differences in the distribution of subtypes between the two types of hosts.

The *gp60* gene sequence of *C. xiaoi* is highly divergent from that of other *Cryptosporidium* spp. Similar to the *gp60* gene of *C. ryanae* (~1548 bp), the *C. xiaoi gp60* gene (~1437 bp) is much longer than those in *C. parvum*, *C. hominis*, and *C. ubiquitum* (~873–1035 bp). Both the nucleotide and amino acid sequences of the *C. xiaoi gp60* gene showed low identity to those of other *Cryptosporidium* spp. This may explain the inability of the commonly used *gp60* primers to amplify DNA of *C. xiaoi* [23]. Similar to *C. ubiquitum*, *C. canis*, *C. felis*, and *C. ryanae*, the trinucleotide repeats of TCA/TCG/TCT encoding a polyserine tract at the 5 end of the *gp60* gene and widely used to differentiate subtypes within subtype families, were absent in the *gp60* sequence of *C. xiaoi* [12,13,17,24]. However, a polyserine tract encoded by AGC/AGT repeats was observed in the *gp60* gene of the subtype family XXIIIl, and subtypes within XXIIIl differed mostly in the number of AGC/AGT repeats. Similar to most *Cryptosporidium* spp., the GP60 protein of *C. xiaoi* has a classic furin cleavage site "RSRR" between GP40 and GP15, which is absent in *C. ubiquitum*, *C. viatorum*, *Cryptosporidium* chipmunk genotype I and skunk genotype [9,11,12,14].

Based on the sequence analysis, the *gp60* gene of *C. xiaoi* displays an extremely high genetic diversity. The analysis of 298 sequences obtained led to the identification of 94 sequence types in 12 subtype families, including significant length polymorphism and sequence variability. The high sequence heterogeneity in this gene, nevertheless, has made PCR amplification difficult, which together with the large amplicon could

be responsible for the poor amplification efficiency. In addition, some samples (13/355) produced double bands in *gp60* PCR, indicating the presence of concurrent infection with different subtypes in sheep and goats. This may facilitate the occurrence of genetic recombination among *C. xiaoi* subtypes, illustrated by the identification of mosaic sequence patterns and 71 potential recombination events in the overall sequence data. Thus, genetic recombination might be responsible for high sequence heterogeneity in the *gp60* gene of *C. xiaoi*. Genetic recombination at the *gp60* locus was observed in *C. parvum*, *C. hominis*, *C. ubiquitum*, and *C. ryanae* [2,12,17].

The *gp60* subtyping results suggest the presence of host adaptation within *C. xiaoi*. Among the 12 subtype families, XXIIIa, XXIIIc, XXIIIg, and XXIIIj were observed only in goats thus far. For the two breeds of goats, Huanghuai goats in Anhui harboured all subtype families of XXIIIa–XXIIIl. In contrast, all 35 samples from Black goats in Guangdong belonged to XXIIIa. The latter could be due to the reduced genetic diversity of *C. xiaoi* in the province. Previously, host-adapted *gp60* subtype families had been identified in other *Cryptosporidium* spp., such as *C. parvum*, *C. hominis*, *C. felis*, *C. ubiquitum*, *C. tyzzeri*, and *C. ryanae* [2,12,17,25–27].

No obvious correlation was found between the distribution of *C. xiaoi* subtype families and geographic locations in this study. Even though all *C. xiaoi* isolates from three farms in Guangdong belonged to XXIIIa, this subtype family was found in goats on two farms in Anhui. Subtyping data of *C. xiaoi* from more geographic locations and diverse animals are needed for better understanding of the distribution of *C. xiaoi* subtypes. Previously, geographical differences had been reported in the subtype distribution of *C. hominis*, *C. parvum*, *C. felis*, *C. ubiquitum*, *C. ryanae*, and *Cryptosporidium* chipmunk genotype I, indicating possible differences in the transmission of these pathogens [9,12,17,25,27,28].
