Molecular Detection of Cryptosporidium spp. and Enterocytozoon bieneusi Infection in Wild Rodents From Six Provinces in China

Enterocytozoon (E.) bieneusi and Cryptosporidium spp. are the most important zoonotic enteric pathogens associated with diarrheal diseases in animals and humans. However, it is still not known whether E. bieneusi and Cryptosporidium spp. are carried by wild rodents in Shanxi, Guangxi, Zhejiang, Shandong, and Inner Mongolia, China. In the present study, a total of 536 feces samples were collected from Rattus (R.) norvegicus, Mus musculus, Spermophilus (S.) dauricus, and Lasiopodomys brandti in six provinces of China, and were detected by PCR amplification of the SSU rRNA gene of Cryptosporidium spp. and ITS gene of E. bieneusi from June 2017 to November 2020. Among 536 wild rodents, 62 (11.6%) and 18 (3.4%) samples were detected as E. bieneusi- and Cryptosporidium spp.-positive, respectively. Differential prevalence rates of E. bieneusi and Cryptosporidium spp. were found in different regions. E. bieneusi was more prevalent in R. norvegicus, whereas Cryptosporidium spp. was more frequently identified in S. dauricus. Sequence analysis indicated that three known Cryptosporidium species/genotypes (Cryptosporidium viatorum, Cryptosporidium felis, and Cryptosporidium sp. rat genotype II/III) and two uncertain Cryptosporidium species (Cryptosporidium sp. novel1 and Cryptosporidium sp. novel2) were present in the investigated wild rodents. Meanwhile, 5 known E. bieneusi genotypes (XJP-II, EbpC, EbpA, D, and NCF7) and 11 novel E. bieneusi genotypes (ZJR1 to ZJR7, GXM1, HLJC1, HLJC2, and SDR1) were also observed. This is the first report for existence of E. bieneusi and Cryptosporidium spp. in wild rodents in Shanxi, Guangxi, Zhejiang, and Shandong, China. The present study also demonstrated the existence of E. bieneusi and Cryptosporidium spp. in S. dauricus worldwide for the first time. This study not only provided the basic data for the distribution of E. bieneusi and Cryptosporidium genotypes/species, but also expanded the host range of the two parasites. Moreover, the zoonotic E. bieneusi and Cryptosporidium species/genotypes were identified in the present study, suggesting wild rodents are a potential source of human infections.


INTRODUCTION
The rodents are one of the largest families of mammals. Wild rodents (e.g., wild rats) are the most widely distributed worldwide. They can shed many pathogens (e.g., Enterocytozoon (E.) bieneusi and Cryptosporidium spp.) into the environment due to living in an open environment, thus becoming potential sources for transmission of pathogens to other animals (Deng et al., 2016;Garcıá-Livia et al., 2020;Gui et al., 2020). In addition, the rodents have a closed relationship with humans. Thus, many pathogens, including E. bieneusi and Cryptosporidium spp., might be transmitted from rodents to humans. (Garcıá-Livia et al., 2020;Gui et al., 2020;Zhao et al., 2020).
E. bieneusi and Cryptosporidium spp. are the common zoonotic enteric pathogens responsible for a majority of parasitic diarrhea diseases worldwide Zhao et al., 2018;Wang S. N. et al., 2020). Both of them can infect humans and a wide variety of animals (e.g., rodents) (Wang et al., 2013;Zhao et al., 2018;Li and Xiao, 2020;Wang S. N. et al., 2020) mainly through water-borne and foodborne routes (Wang et al., 2013;Zhao et al., 2018). In general, healthy people infected with both pathogens are asymptomatic or manifest symptoms of self-limiting diarrhea. However, the infection of E. bieneusi and Cryptosporidium spp. in immunocompromised individuals may cause chronic or lifethreatening diarrheas (Wang et al., 2013;Sutthikornchai et al., 2021). Owing to their significance in public health, Cryptosporidium spp. and E. bieneusi have been put into Category B Priority Pathogen list by the National Institute of Allergy and Infectious Diseases (NIAID) (NIAID, 2018). Moreover, E. bieneusi is also listed on the Environmental Protection Agency (EPA) microbial contaminant candidate list of concern for waterborne transmission (Didier et al., 2009).
In view of such severe situations, it is essential to investigate the prevalence of E. bieneusi and Cryptosporidium spp. in different rodent species and identify their species/genotypes. However, information regarding Cryptosporidium spp. infection in rodents was limited in China, which was only reported in Microtus fuscus (Qinghai vole) and Ochotona curzoniae (wild plateau pika) in Qinghai , brown rats (Rattus norvegicus) in Heilongjiang , bamboo rats in Sichuan (Liu et al., 2015), pet chinchillas in Beijing, Henan and Guizhou , commensal rodents in Henan and Fujian (Zhao et al., 2015), brown rats in Heilongjiang , wild, laboratory, and pet rodents in Beijing, Henan, Fujian and Sichuan (Lv et al., 2009), bamboo rats in Guangdong, Hunan, Guangxi, Jiangxi and Hainan (Wei et al., 2019;Li et al., 2020a;Li et al., 2020b), Asian house rats, brown rats, Edward's long-tailed rats and muridae in Hainan . In China, E. bieneusi in rodents has been only reported in Heilongjiang , Beijing , Henan Wang J. et al., 2020), Guizhou , Sichuan (Deng et al., 2016), Shandong , Guangdong , Hunan Gui et al., 2020), Jiangxi , Chongqing , Guangxi , and Hainan .
However, it is still not known whether E. bieneusi and Cryptosporidium spp. are carried by wild rodents in Shanxi, Guangxi, Zhejiang, Shandong, and Inner Mongolia, China. Thus, the present study was performed to estimate the prevalence and genotypes of E. bieneusi and Cryptosporidium spp. in wild rodents by the molecular biological method.

Specimen Collection
A total of 536 feces samples were collected from four rodent species from Daqing City in Heilongjiang (n = 41; 39 S. dauricus, 2 R. norvegicus), Taigu County in Shanxi (n = 53, R. norvegicus), Nanning City in Guangxi (n = 74, M. musculus), Weihai City in Shandong (n = 227, R. norvegicus), Jiaxing City in Zhejiang (n = 119, R. norvegicus) and Xilingol League in Inner Mongolia (n = 22, L. brandti), China from June 2017 to November 2020. These rodents were captured by trapping method. The rodents had been euthanized by CO2 inhalation, and then the fresh feces sample (approximately 500 mg) was collected directly from the intestinal and rectal content of each rodent, and then was placed into ice boxes and sent to the laboratory. Information regarding sampling time, region, and species was recorded. This study was approved by the Ethics Committee of Qingdao Agricultural University.

DNA Extraction and PCR Amplification
Genomic DNA was extracted from fecal sample of approximately 200 mg using the E.Z.N.A. ® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer's instructions, and then was stored at -20°C prior to PCR. The prevalence and genotypes of E. bieneusi were identified by PCR amplification of the ITS gene according to the previous description . Cryptosporidium spp. in the fecal samples was confirmed by PCR amplification of the SSU rRNA gene according to the previous report . The positive and negative controls were included in each test. The secondary PCR products were observed using UV light after an electrophoretic analysis at a 1.5% agarose gel containing ethidium bromide.

Sequence and Phylogenetic Analyses
The positive PCR specimens were sent to Sangon Biotech Company (Shanghai, China) for sequencing. A new PCR product should be sequenced if previously produced sequences had single nucleotide substitutions, insertions or deletions. The nucleotide sequences were aligned and analyzed with reference sequences by using the Clustal X 1.83 program and Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/), in order to determine the species/genotypes of Cryptosporidium spp. and E. bieneusi. The phylogenetic trees were reconstructed with Mega 5.0 using neighbor-joining (NJ) method under Kimura 2parameter model (1,000 replicates). All nucleotide sequences were deposited in GenBank with accession numbers MT647749 -MT647806 and OK117929 -OK117932 for E. bieneusi, and MT561508 -MT561533 for Cryptosporidium spp.

Statistical Analysis
The statistical analysis for the prevalence of E. bieneusi and Cryptosporidium in wild rodents from different region, season, sampling year, and species were performed by using c2 test in SAS version 9.1 (SAS Institute, Cary, NC, USA). The results were considered to be statistically significant when P < 0.05. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were also calculated to compare the magnitude of various risk factors for E. bieneusi and Cryptosporidium prevalence.
E. bieneusi and Cryptosporidium spp. coinfection was found in three wild rodents in this study. All of them were R. norvegicus collected in 2018. Two were collected from Zhejiang Province, and the remaining one was collected from Shanxi Province.
Distribution of Cryptosporidium spp. and E. bieneusi Cryptosporidium sp. rat genotype II/III, Cryptosporidium felis, and Cryptosporidium viatorum were identified in the investigated rodents through the analysis of SSU rRNA gene of Cryptosporidium. Furthermore, two Cryptosporidium genotypes with uncertain species status were observed ( Figure 1 and Table 1). Cryptosporidium sp. novel1 and C. felis were found in S. dauricus in Heilongjiang. C. viatorum and Cryptosporidium sp. rat genotype II/III were only identified in M. musculus in Guangxi. Cryptosporidium sp. novel2 was found in three provinces Zhejiang (R. norvegicus), Shanxi (R. norvegicus), and Guangxi (M. musculus) ( Table 1).
More than 30 Cryptosporidium species/genotypes have been identified in rodents. However, only five species/genotypes were identified in this study, including C. viatorum, C. felis, Cryptosporidium sp. rat genotype II/III, Cryptosporidium sp. novel1, and Cryptosporidium sp. novel2. Among them, Cryptosporidium sp. rat genotype II/III, previously reported in rodents (Garcıá-Livia et al., 2020;Jezǩováet al., 2021), was also identified in this study, which was further confirmed that Cryptosporidium sp. rat genotype II/III was one of the prevalent Cryptosporidium genotypes in rodents. Moreover, two uncertain species of Cryptosporidium (Cryptosporidium sp. novel1 and novel2) were also identified in this study. Cryptosporidium sp. novel1 (isolates 251, 261, 263, and 265) was grouped into a new separate clade. Cryptosporidium sp. novel2 (isolates 32, 44, 63, 67, 70, 155, and 245), grouped with Cryptosporidium environmental. The results indicate two new genotypes/species that have clustered a branch in phylogenetic analysis with environmental isolates of Cryptosporidium spp.   One of the reasons that in environmental samples, it is difficult to determine the species and genotype is the simultaneous contamination of several species and genotypes in samples that after sequencing cannot detect a known species or genotype. Unfortunately, other genes such as COWP and HSP70 of the uncertain species have also not been successfully amplified. Thus, the investigation should be continue performed to further confirm whether presence of the two uncertain species of Cryptosporidium in wild rodents. C. viatorum, has been identified in humans Lebbad et al., 2013;Adamu et al., 2014;Ayinmode et al., 2014;De Lucio et al., 2016;Sanchez et al., 2017;Ukwah et al., 2017;Sannella et al., 2019). C. viatorum was first found in travellers who returned to the United Kingdom from the Indian subcontinent, with clinical signs of diarrhea, fever, headache, abdominal pain, nausea, vomiting, and marked weight loss (Elwin et al., 2012). So far, C. viatorum has been documented in the following countries: Bangladesh, Ethiopia, Barbados, Kenya, Colombia, Nigeria, Pakistan, Guatemala, India, and Nepal (Insulander et al., 2013;Lebbad et al., 2013;Adamu et al., 2014;Ayinmode et al., 2014;De Lucio et al., 2016;Sanchez et al., 2017;Ukwah et al., 2017;Sannella et al., 2019). Besides, C. viatorum was also found in China, such as Hainan Province (Leopoldamys edwardsi), Guangdong Province (Berylms bowersi), and Chongqing City (Leopoldamys edwardsi) in China, and in Australia (Rattus lutreolus) Chen et al., 2019;Zhao et al., 2019). C. felis has been widely reported in cats , in addition to patients with HIV/AIDS in Peru, Ethiopia, Nigeria, Jamaica, and Portugal (Cama et al., 2003;Jiang et al., 2020). In this study, C. viatorum and C. felis were found in M. musculus and S. dauricus, which was worth for further research, e.g., whether wild rodents are potentially important reservoirs for C. viatorum and C. felis transmission to humans. More importantly, this is the first study showing existence of Cryptosporidium spp. in S. dauricus, which has expanded the host ranges of Cryptosporidium. At present, more than 400 genotypes of E. bieneusi have been identified, most of which exhibit host specificity (Santıń and Fayer, 2011;Wang S. N. et al., 2020). At least 48 genotypes of E. bieneusi infect both human and animals, bringing zoonoses risks (Li and Xiao, 2019). Through phylogenetic analysis, these genotypes were divided into at least 11 groups, e.g., Group 1 to Group 11 Wang S. N. et al., 2020). To date, some genotypes were found in rodents, of which 15 genotypes (CZ3, Peru6, BEB6, C, D, EbpA, EbpC, H, Peru8, Peru11, Peru16, PigITS5, S6, IV, and Nig7) were reported to infect human. In China, EbpA, EbpC, CHY1, N, D, Peru11, S7, SCC-2, PGP, Peru6, J, PigEBITS7, BR1 and BR2, Type IV, Peru8, ESH02, CHG5, HNR-I to HNR-VII, K, CQR-1, CQR-2, CQR-3, GDR-1, GDR-2, GDR-3. SCC-1, SCC-3, SCC-4, CHY1, Nig7 CHG9, ChG14, BEB6, CHG2, SC02, CE01 and CE02 genotypes were reported in rodents (Feng et al., 2009;Zhao et al., 2018;Wang et al., 2019;Li J. et al., 2020;Wang J. et al., 2020;Zhao et al., 2020). However, only 5 known genotypes (XJP-II, EbpC, EbpA, D, and NCF7) and 11 novel genotypes (ZIR1 to ZJR7, GXM1, HLJC1, HLJC2, and SDR1) were identified in the present study. Among them, 14 genotypes were clustered into a highly-supported monophyletic clade (Group 1), indicating that these genotypes are human-pathogenic types and may cause infection between humans and rodents, thus becoming a public health significance. This was the first record of E. bieneusi in S. dauricus. Eleven novel genotypes (ZIR1 to ZJR7, GXM1, HLJC1, HLJC2, and SDR1) were recorded in rodents for the first time. Of which, ZJR1, ZJR3, ZJR4, ZJR5, ZJR6, ZJR7, SDR1, HLJC1, and GXM1 were grouped into Group 1 (Figure 2), thus suggesting that rodents (R. norvegicus, M. musculus, and S. dauricus) may play an important role in the transmission of E. bieneusi between rodents and humans. Genotype XJP-II was previously found in pigs in Xinjiang (Li D. F. et al., 2019b), and NCF2 was also identified in farmed foxes (Vulpes lagopus) Ma et al., 2020) and raccoon dogs (Nyctereutes procyonoides) (Xu et al., 2016) in China, Kangaroo in Australia . Genotypes EbpC, EbpA, and D were frequently found in humans and a broad range of animals (Wang et al., 2013;Liu et al., 2017;Qi et al., 2018;Zou et al., 2018;Yu et al., 2020). The results showed that natural transmission of E. bieneusi among rodents, humans and many other animals may occur. More importantly, the three ITS genotypes were also found in water in China, which should be paid more attention to prevent the water-borne transmission of E. bieneusi (Hu et al., 2014).
Collectively, the present study firstly demonstrated that existence of Cryptosporidium spp. (3.4%, 18/536) and E. bieneusi (11.6%, 62/536) in rodents in Shanxi, Guangxi, and Zhejiang, China. Three known Cryptosporidium species/genotypes (C. viatorum, C. felis, and Cryptosporidium sp. rat genotype II/III), two uncertain Cryptosporidium species/genotypes (Cryptosporidium sp. novel1 and Cryptosporidium sp. novel2), 5 known E. bieneusi genotypes (XJP-II, EbpC, EbpA, D, and NCF7) and 11 novel E. bieneusi genotypes (ZJR1 to ZJR7, GXM1, HLJC1, HLJC2, and SDR1) were identified in the investigated rodents, suggesting rodents can act as a potential source of human and animal infections. E. bieneusi was more prevalent in R. norvegicus, whereas Cryptosporidium spp. was more frequently identified in S. dauricus. The present study also demonstrated that S. dauricus was the host of E. bieneusi and Cryptosporidium spp. for the first time. This study expanded the host range of these two parasites, which not only provided basic data for distribution of E. bieneusi and Cryptosporidium genotypes/species, but also provided foundation data for the prevention and control of E. bieneusi and Cryptosporidium spp. in China.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/genbank/, MT647749-MT647806, OK117929-OK117932, MT561508-MT561533.

ETHICS STATEMENT
This study was approved by the Ethics Committee of Qingdao Agricultural University.