Prevalence and molecular characterization of Cryptosporidium spp. in Père David’s deer ( Elaphurus davidianus ) in Jiangsu, China

siyang.huang@hotmail.com Abstract Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père David’s deer. In this study, 137 fecal samples from Père David’s deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA ( SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein ( gp60 ) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père David’s deer in this These results indicate that the Cryptosporidium isolated from the Père David’s deer in this study were close to the Cryptosporidium deer genotype. This study reported the prevalence (1.46%, 2/137) of Cryptosporidium infection in Père David’s deer in the Dafeng Reserve, China, for the first time. The genotype identified in Père David’s deer in the Dafeng Reserve was Cryptosporidium deer genotype with 3 mutants, which was closely related to C. ryanae and C. bovis . Further investigation into the transmission dynamics of these pathogens should be continued.


Introduction
Cryptosporidiosis is caused by Cryptosporidium spp., which is an important enteric apicomplexan parasite of zoonosis in the world (Parsons et al., 2015;Tanriverdi et al., 2007;Zhang et al., 2016). It is a critical emerging infectious disease in humans and animals that can lead to diarrhea or other serious symptoms (Zhao et al., 2015). In general, cryptosporidiosis is transmitted through the fecal-oral route, when ingesting food or water contaminated with infective oocysts. Currently, there is no effective drug or vaccine to cure or prevent cryptosporidiosis. Therefore, this disease has caused significant economic losses in animal husbandry. In addition, infected animals may be a source of secondary infection, because they can serve as potential carriers for human and other animal infections via excreting feces, including oocysts, that contaminate food and water (Deng & Cliver, 1999).
Père David's deer (Elaphurus davidianus), also called Milu deer, native to the Yangtze River Basin of China, is an endangered deer species in the world and listed as Extinct in the Wild by the International Union for Conservation of Nature (IUCN). It became extinct in the wild in China at the end of the 19th century. Fortunately, from 1985 to 1987, two groups of 40 and 39 Père David's deer were reintroduced to China from the UK and raised in the Nanhaizi Nature Reserve and Dafeng Reserve, respectively. The largest population in the world lives in the Dafeng Reserve, which is historically synonymous with Père David's deer (Ding et al., 2018).

Specimen collection and preparation
A total of 137 fecal samples of Père David's deer were collected between July 2017 and August 2018 in the Dafeng Reserve, Jiangsu Province, China. The samples were collected immediately after excreted onto the ground using sterile gloves and placed in individual plastic bags. No visible clinical signs were observed in these deer. The samples were pretreated in the laboratory in the following steps: 50 g of feces were placed in a beaker, diluted with normal saline, and stirred evenly with a glass rod. Then, the suspension was filtered with a 200-mesh sieve. The filtrate was loaded into a 50-mL centrifuge tube, centrifuged at 3,000× g for 10 min, and the precipitate was collected and stored at -20°C for further study.

DNA extraction and PCR amplification
Genomic DNA was extracted from each sample using the E.Z.N.A. Stool DNA Kit (OMEGA, USA) according to the manufacturer's directions and stored at -20°C or immediately used for PCR. Cryptosporidium species and genotypes were examined by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, as previously described (Zhao et al., 2013). For further identification and subtype detection, the samples positive for SSU rRNA were further analyzed by nested-PCR targeting the 60-kDa glycoprotein (gp60) gene (Alves et al., 2003;Feng et al., 2012;Li et al., 2014). The cycling conditions were as follows: 5 min at 95°C, followed by 35 cycles, each composed of 45 s at 94°C, an annealing step at a suitable temperature (Table 1) for 45 s, and 1 min at 72°C, and the final extension at 72°C for 10 min. Positive and negative controls were included in each reaction. The products were observed under UV light after electrophoresis in 1.5% agarose gels stained with ethidium bromide.

Sequencing and phylogenetic analyses
All PCR products were sequenced by the GenScript Company (Nanjing, China). The sequence accuracy was confirmed by bidirectional sequencing. To determine Cryptosporidium species and subtypes, the sequencing results were aligned with known reference sequences of Cryptosporidium available in GenBank using BLAST . MEGA 5.0 was used to construct the phylogenetic trees using neighbor-joining (NJ) analysis of the SSU rRNA sequences, based on the Kimura-2-parameter model, and bootstrapping was performed using 1000 replicates. The nucleotide sequences obtained in this study were deposited in the GenBank under accession number MK571183.

Prevalence of Cryptosporidium
In this study, 2 of 137 fecal samples were positive for Cryptosporidium infection. The overall prevalence of Cryptosporidium was 1.46% in Père David's deer in the Dafeng Reserve. The result was similar to 3.7% seen in wild red deer, European leisure deer, white-tailed deer and mouflon sheep in the Czech Republic (Kotkova et al., 2016), but lower than that in the red deer, Père David's deer and sika deer in Henan and Jilin, China (6.8%), the Hokkaido sika deer in Japan (7.8%) and white-tailed deer in central Maryland (12.5%) (Huang et al., 2018;Kato et al., 2016;Santin & Fayer, 2015). Although one study indicated that Cryptosporidium was found in Père David's deer, there was no detailed information about prevalence. Thus, it is difficult to compare the prevalence with that in other studies. In addition, due to the influence of ecological conditions, age distributions, seasons, management systems, sample sizes and other factors, explaining the discrepancies in the prevalence of Cryptosporidium among different studies is challenging (Huang et al., 2014).

Cryptosporidium species and genotypes
Two Cryptosporidium-positive samples were sequenced and genotyped by the sequence analysis of the SSU rRNA gene. According to the results of the BLAST (NCBI) analysis, both isolates represented the Cryptosporidium deer genotype. The Cryptosporidium deer genotype (GenBank accession numbers: KX259129), which was recently reported in red deer in Henan and Jilin, China, has two nucleotide deletions (-/G position 8, -/T position 16) and one nucleotide substitution (G/T position 11) (Huang et al., 2018) in the two isolates in the present study. In the Cryptosporidium genome, the gp60 gene was used for C. parvum and C. ubiquitum subtype analysis due to its heterogeneity and biological correlation. Although no C. parvum and C. ubiquitum were detected, the two positive samples were analyzed by nested-PCR targeting the gp60 gene (Feng et al., 2007b). The results were negative; no PCR amplicon was amplified. Currently, there are several reports on cervid infections with the Cryptosporidium deer genotype in England, Australia, Czech Republic, China, and Japan (Cinque et al., 2008;Feng et al., 2007a;Koehler et al., 2016;Perz & Le Blancq, 2001;Robinson et al., 2011;Xiao et al., 2002). However, there is little genotype information about Cryptosporidium in Père David's deer. In the present study, the genotype identified in the Père David's deer Table 1. Primers used in the study, annealing temperatures used in the PCR, and expected sizes of the PCR products.

Phylogenetic analyses
Phylogenetic relationships were established by the NJ method; the Plasmodium cathemerium sequence was used as the outgroup, and the sequence similarity between Cryptosporidium species and genotypes available in GenBank was observed based on SSU rRNA (Figure 1). Cryptosporidium forms two main groups, one of which includes C. muris, C. serpentis, C. galli and C. andersoni, previously known as parasitic gastrosporidium. The other group includes C. bovis, C. ryanae, C. scrofarum, Cryptosporidium pig genotype, C. avium, C. baileyi, C. canis, C. suis, C. lemurs, C. wrairi, C. meleagridis, C. parvum, Cryptosporidium deer genotype, and the isolated strain of Cryptosporidium derived from Père David's deer (MK571183). The results indicated that Cryptosporidium spp. Père David's deer (the newly generated sequences in this study) was clustered in the Cryptosporidium deer genotype branch. The genotype shares a branch with isolates from the United States, Japan and China and is closely related to C. ryanae and C. bovis. These results indicate that the Cryptosporidium isolated from the Père David's deer in this study were close to the Cryptosporidium deer genotype. This study reported the prevalence (1.46%, 2/137) of Cryptosporidium infection in Père David's deer in the Dafeng Reserve, China, for the first time. The genotype identified in Père David's deer in the Dafeng Reserve was Cryptosporidium deer genotype with 3 mutants, which was closely related to C. ryanae and C. bovis. Further investigation into the transmission dynamics of these pathogens should be continued.