Occurrence and genetic characteristics of Cryptosporidium spp. and Enterocytozoon bieneusi in pet red squirrels (Sciurus vulgaris) in China

Cryptosporidium spp. and Enterocytozoon bieneusi are two well-known protist pathogens which can result in diarrhea in humans and animals. To examine the occurrence and genetic characteristics of Cryptosporidium spp. and E. bieneusi in pet red squirrels (Sciurus vulgaris), 314 fecal specimens were collected from red squirrels from four pet shops and owners in Sichuan province, China. Cryptosporidium spp. and E. bieneusi were examined by nested PCR targeting the partial small subunit rRNA (SSU rRNA) gene and the ribosomal internal transcribed spacer (ITS) gene respectively. The infection rates were 8.6% (27/314) for Cryptosporidium spp. and 19.4% (61/314) for E. bieneusi. Five Cryptosporidium species/genotypes were identified by DNA sequence analysis: Cryptosporidium rat genotype II (n = 8), Cryptosporidium ferret genotype (n = 8), Cryptosporidium chipmunk genotype III (n = 5), Cryptosporidium rat genotype I (n = 4), and Cryptosporidium parvum (n = 2). Additionally, a total of five E. bieneusi genotypes were revealed, including three known genotypes (D, SCC-2, and SCC-3) and two novel genotypes (RS01 and RS02). Phylogenetic analysis revealed that genotype D fell into group 1, whereas the remaining genotypes clustered into group 10. To our knowledge, this is the first study to report Cryptosporidium spp. and E. bieneusi in pet red squirrels in China. Moreover, C. parvum and genotype D of E. bieneusi, previously identified in humans, were also found in red squirrels, suggesting that red squirrels may give rise to cryptosporidiosis and microsporidiosis in humans through zoonotic transmissions. These results provide preliminary reference data for monitoring Cryptosporidium spp. and E. bieneusi infections in pet red squirrels and humans.

E. bieneusi have been recognized as category B pathogens by the National Institutes of Health due to their ease of transmission in spite of low mortality 9 .
To detect and evaluate potential zoonotic transmissions, it is necessary to accurately distinguish Cryptosporidium spp. and E. bieneusi on the molecular level 10 . To date, at least 37 species and over 70 genotypes of Cryptosporidium spp. have been described 11 . Among them, 11 Cryptosporidium species have been identified in rodents, Cryptosporidium parvum and C. muris are the most common 12 . For E. bieneusi, more than 474 genotypes have been identified based on the internal transcribed spacer (ITS) region of the rRNA gene 13 , and more than 35 genotypes have been determined in rodents 14 . These genotypes can be classified into eleven groups (groups 1-11) by phylogenetic analyses 13 . Group 1 comprises the majority of zoonotic potential genotypes, whereas the remaining (groups 2-11) are considered as the host-adapted groups, which are mostly found in specific hosts or water 13 .
In China, Cryptosporidium spp. and E. bieneusi have been detected in a wide range of hosts, including carnivores, lagomorphs, primates, birds, and rodents 14,15 . Pet rodents, in particular (e.g. chinchillas, red-bellied tree squirrels, guinea pigs, and chipmunks), are considered potential sources of Cryptosporidium spp. and E. bieneusi infections in humans [16][17][18][19] . The red squirrel (Sciurus vulgaris) is a popular pet in China, which is widely bred in pet shops and homes for its appearance and mild-mannered nature. However, there is no published data regarding the prevalence of Cryptosporidium spp. and E. bieneusi in pet red squirrels, and the role of the red squirrels in the transmission of the two pathogens remains poorly investigated. Thus, we examined the occurrence of Cryptosporidium spp. and E. bieneusi in red squirrels, and evaluated their potential role in the zoonotic transmission of human cryptosporidiosis and microsporidiosis.

Phylogenetic relationship of E. bieneusi.
A phylogenetic analysis of the ITS gene sequences of all the genotypes of E. bieneusi obtained here and reference genotypes published previously revealed that genotype D clustered in group 1 and further clustered into 1a, whereas genotypes SCC-2, SCC-4, and two novel genotypes (RS01 and RS02) clustered in group 10 ( Fig. 2).
Previous studies have indicated that five Cryptosporidium species and nine Cryptosporidium genotypes exist in various rodents in China (Table 3) 12,22,23 . In this study, five different Cryptosporidium species/genotypes were identified, including Cryptosporidium rat genotypes I and II, Cryptosporidium ferret genotype, Cryptosporidium chipmunk genotype III, and C. parvum. Cryptosporidium rat genotypes I and II have been found in brown rats in the Philippines 34 , Nigeria 20 , Australia 35 , and China 22 , even in South Nation River watershed, raw wastewater, and environmental samples in the United Kingdom, Canada, and China [36][37][38] . Cryptosporidium ferret genotype has been found in ferrets and red squirrels in Italy 39 . The Cryptosporidium chipmunk genotype III was previously reported in red squirrels, eastern squirrels, eastern chipmunks, and deer mice in the USA 40 . To date, little is known regarding the disease-causing potential of the four genotypes in humans and livestock; thus bringing attention to the need for epidemiological molecular surveillance of Cryptosporidium spp. for the assessment of infectivity across different hosts.
C. parvum is one of the two predominant Cryptosporidium species in humans 41 . C. parvum has been identified in humans in Henan province, China 42 . Moreover, C. parvum infections have been observed in brown rats in Japan, mice and red-backed voles in the USA, brown rats in Iran, striped field mice in Slovak Republic, and hamsters, Siberian chipmunks, chinchillas, and Bamboo rats in China, highlighting the prevalence of C. parvum in www.nature.com/scientificreports www.nature.com/scientificreports/ rodents (Table 3) 12,25,27 . In addition, C. parvum has also been found in other animals, such as cattle, sheep, goats, deer, alpacas, horses, dogs, gray wolves, raccoon dogs, cats, and pigs 43,44 . In this study, only two C. parvum isolates were identified in investigated red squirrels; however, these isolates may result in emerging zoonotic infections through the oral-fecal route.
Three known genotypes (D, SCC-2, and SCC-3) and two novel genotypes (RS01 and RS02) were identified in this study. Genotype D was the predominant genotype (44.3%, 27/61). This finding was similar to previous reports in mice in Czech Republic and Germany 45 (32.3%, 10/31), mice in Poland 46 (33.3%, 10/30), and brown www.nature.com/scientificreports www.nature.com/scientificreports/ rats (89.5%, 17/19) and red-bellied tree squirrels (75%, 18/24) in China 12,18 . In China, genotype D has been identified in humans and various animals, such as nonhuman primates, cattle, sheep, horses, pigs, dogs, cats, and in wastewater 14,[47][48][49] . This study demonstrated the presence of genotype D in red squirrels for the first time, suggesting that red squirrels could play a potential role in the disease dissemination of E. bieneusi to humans. conclusions This is the first report on the incidence of Cryptosporidium spp. and E. bieneusi in pet red squirrels in China. The infection rates of Cryptosporidium spp. and E. bieneusi were 8.6% and 19.4%, respectively. The detection of zoonotic C. parvum and genotype D of E. bieneusi suggests that red squirrels are a potential source of cryptosporidiosis and microsporidiosis in humans. However, the infection sources and transmission dynamics between red squirrels and humans remain unknown, thus emphasizing on the importance of further follow-up studies of the transmission dynamics of these pathogens.

Materials and Methods
ethics statement. The present study protocol was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of Sichuan Agricultural University, and all methods were performed in accordance with the relevant guidelines and regulations. Permission was obtained from the owners or shop managers before the fecal specimens were collected. collection of specimens. A total of 314 fecal specimens were collected from red squirrels from four pet shops (n = 269) and owners (n = 45) in the Sichuan province, southwestern China between September 2016 and December 2017 (Table 1). All tested pet shops only raised red squirrels and served as suppliers of red squirrels to other pet shops. Sample size was approximately 20% of the squirrels from each shop, and small-scale shops (population less than 50) were not included. The four pet shops are distributed in Jianyang (104°32′E, 30°24′N), Pengzhou (103°57′E, 30°59′N), Wenjiang (103°51′E, 30°40′N), and Jintang (104°24′E, 30°51′N). Pet squirrels from owners were primarily distributed around the urban areas of Chengdu city (104°03′E-104°08′E, 30°36′N-30°52N′). In both pet shops and homes, red squirrels were housed in separate cages. Approximately 30-50 g fresh fecal samples were collected from the bottom of each cage after defecation using a sterile disposal latex glove and then immediately placed into individual disposable plastic bags. No obvious clinical signs were observed at the time of sampling, and the age, sex, and source were recorded at the same time. All fecal specimens were stored in 2.5% potassium dichromate solution at 4 °C until processing. DNA extraction. The fecal specimens were washed three times in distilled water with centrifugation at 3,000 × g for 10 min to remove the potassium dichromate. Genomic DNA was extracted from approximately 200 mg of each processed fecal specimen using an E.Z. N. A. R Stool DNA kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer's recommended instructions. The extracted DNA was stored at −20 °C until molecular analysis.
Genotyping of Cryptosporidium spp. and E. bieneusi. Cryptosporidium spp. were identified by nested polymerase chain reaction (PCR) amplification of an SSU rRNA gene fragment of ~830 bp designed by Xiao et al. 50 . E. bieneusi genotypes were determined by nested PCR amplification of a 392-bp fragment containing the entire ITS (243 bp) and portions of the flanking large and small subunits of the rRNA gene 31  www.nature.com/scientificreports www.nature.com/scientificreports/ E. bieneusi) and negative control with no DNA added were included in all PCR assays. The secondary PCR products were examined by agarose gel electrophoresis and visualized after ethidium bromide staining. Sequence analysis. All nested PCR positive-products were sequenced using the same PCR primers as those used for the secondary PCRs on an ABI 3730 instrument (Applied Biosystems, Foster City, CA, USA) at the BioSune Biotechnology Company (Shanghai, China). The nucleotide sequences of each obtained gene were aligned and analyzed using the Basic Local Alignment Search Tool and Clustal X (http://www.clustal.org/) with reference sequences retrieved from GenBank to identify Cryptosporidium spp. and E. bieneusi genotypes.

Phylogenetic analyses.
To support the Cryptosporidium species/genotypes and assess the genetic relationships between the E. bieneusi genotypes in the present study and reference sequences previously published in GenBank, phylogenetic analysis was performed using Phylip version 3.69 package and by constructing a neighboring-joining tree using Mega 6 software (http://www.megasoftware.net/), which is based on evolutionary distances calculated using a Kimura 2-parameter model. The MegAlign program in the DNA Star software package (version 5.0) was used to determine the degree of sequence identity. The reliability of these trees was assessed using bootstrap analysis with 1,000 replicates.

Statistical analysis.
Variations in the occurrence of Cryptosporidium spp. (y1) and E. bieneusi (y2) in red squirrels according to age (x1), sex (x2), and geographical location (x3) were analyzed by χ 2 test using SPSS V20.0 (IBM, Chicago, IL, USA). Each of these variables was included in the binary logit model as an independent variable by multivariable regression analysis. When the P value was less than 0.05, the results were considered statistically significant. The adjusted odds ratio (OR) and 95% confidence interval (CI) for each variable were calculated with binary logistic regression, and all risk factors were entered simultaneously.

GenBank accession numbers.
Representative nucleotide sequences were deposited in GenBank with the following accession numbers: MH940281-MH940290.

Data availability
All data generated or analysed during this study are included in this published article and its Supplementary Information Files.