Molecular Characterization of Cryptosporidium spp. in Brandt's Vole in China
Shengyong Feng
Han Chang
Ye Wang1 
Hongxuan He- 1National Research Center for Wildlife Borne Diseases, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
Cryptosporidium spp. are important intestinal parasites that infect humans and various animals, including wildlife. Currently, few epidemiological data in wild rodents, especially in voles, are available. In the present study, a total of 678 Brandt's vole feces samples were collected from Maodeng Livestock Farm and East Ujimqin, Inner Mongolia. The overall prevalence of Cryptosporidium spp. was 18.7%. Significant differences were not found between genders but between locations and weight groups. Moreover, three known species/genotypes, C. suis, Cryptosporidium environmental sequence and muskrat genotype II, and a novel Cryptosporidium species/genotypes of Brandt's vole was identified. To the best of our knowledge, this is the first report of Cryptosporidium spp. infection in Brandt's vole worldwide. These findings imply Brandt's voles might be a potential source of human cryptosporidiosis.
Introduction
Cryptosporidiosis, caused by species of the genus Cryptosporidium, is one of the common etiologies of diarrhea in humans and animals (1). The oocysts shed from infected hosts can survive for quite a long time in the environment (2). Thus, infection is more likely to occur by ingesting water or foods contaminated with oocysts (3). The prognosis of cryptosporidiosis may be chronic infection or life-threatening in certain people (4).
Rodents can carry a large number of pathogens, including bacteria, viruses and parasites, which pose a threat to public health (5). Brandt's vole (Lasiopodomys brandtii) is a small, non-hibernating, herbivorous rodent species, and mainly distributed in the grasslands of Inner Mongolian of China, Mongolia, and Southeast Baikal region of Russia (6). It is generally agreed that Brandt's vole is one of the important grassland pests due to their damage to grasslands (6).
Currently, Cryptosporidium has been identified from domestic mammals (7), birds (8), reptiles (9), amphibians (10), and fishes (11). More than 40 species of Cryptosporidium have been identified (7). However, it is rarely reported in wild rodents, especially in voles. Here we reported the prevalence of Cryptosporidium spp. in wild Brandt's vole from Inner Mongolian, China. Data from this study contributes to enriching the epidemiological data of Cryptosporidium in China.
Materials and Methods
Ethics Statement
This study was conducted in accordance with the Guidelines for the Care and Use of Animals in Research, which are issued by the Institute of Zoology, Chinese Academy of Sciences. This work was reviewed and approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences.
Sample Collection and DNA Extraction
From 2017 to 2018 (August to September each year), Brandt's voles were trapped using live traps baited with peanuts (12) at two discontinuous habitats, Maodeng Livestock Farm (MD) and East Ujimqin (DWQ), of Xilingol Grassland, Inner Mongolia, China. The climatic conditions between DWQ and MD are similar. However, MD is experiencing more anthropogenic disturbances, such as village and grazing activity, compared with DWQ (13). Sampling was conducted before 10 a.m. and after 5 p.m. Fecal samples were collected into 2 ml sterilized centrifuge tubes from each trap. The sex, weight and reproductive status of the captured Brandt' voles were recorded. The trapped vole was released after recording the individual details. The tubes were marked, put into a box filled with ice packs and transported to a refrigerator as soon as possible.
The total genomic DNA was extracted from 200 mg feces with the EZNA® Stool DNA Kit (Omega Biotek Inc., Norcross, USA) following the manufacturer's instructions. The purified DNA was stored at −20°C for further PCR. Cryptosporidium species/genotypes were determined by amplifying the SSU rRNA gene under nested PCR according to the previous studies (14, 15). Positive control and negative control are added in each amplification. The secondary PCR products were visualized by 2% agarose gel electrophoresis containing GoldView™ (Solarbio, China) stained.
Sequencing and Phylogenetic Analyses
Positive secondary PCR products were bi-directionally sequenced by the Sino Geno Max Company (Beijing, China). Chromatograms of the forward and reverse sequences were manually confirmed and assembled with Lasergene SeqMan software (DNASTAR, Madison, Wisconsin, USA). Cryptosporidium species/genotypes were determined by aligning with reference sequences available in GenBank database with the ClustalX 1.83 software package. Phylogenetic relationship of Cryptosporidium spp. was constructed under MEGA 7.0 (16) with the Neighbor-joining algorithm in Jukes-Cantor method (17), and the robustness of clusters was estimated using a bootstrap of 1, 000 replicates (18).
Statistical Analysis
Differences in infection rates were compared with the chi-square test under SPSS 19.0 (SPSS Inc., Chicago, USA). Differences were considered to be statistically significant when P < 0.05.
Results
Prevalence of Cryptosporidium Spp. in Brandt's Voles
In total, 678 Brandt's voles were sampled from DWQ and MD of Inner Mongolia Autonomous Region, China. 127 samples (18.7%) were found to be Cryptosporidium-positive by testing the partial small subunit (SSU) rRNA gene via PCR. The infection rates of Cryptosporidium spp. in these two regions were 15.6 and 23.6% (χ2 = 6.845, df = 1, P = 0.009), respectively. The prevalence of Cryptosporidium spp. in female Brandt's voles (18.9%) was quite similar to that in male Brandt's voles (18.5%) (χ2 = 0.018, df = 1, P = 0.893). The infection rate of voles weighing <25 g was significantly higher than those weighing between 25 and 35 g and those weighing more than 35 g (χ2 = 17.753, df = 2, P = 0.000) (Table 1).
Table 1. Prevalence and distribution of Cryptosporidium species/genotypes in Brandt' voles in Inner Mongolia, China.
Cryptosporidium Species/Genotypes
Of the 127 PCR positive samples, 122 were sequenced successfully. Furthermore, 17 representative sequences were obtained through sequence analysis. Four Cryptosporidium species/genotypes of Brandt's voles were identified by aligning against the reference Cryptosporidium sequences and constructing phylogenetic tree with the SSU rRNA gene sequences (Figure 1), including three known species, C. suis, Cryptosporidium environmental sequence and Cryptosporidium muskrat genotype II, and one novel Cryptosporidium genotypes, termed Cryptosporidium Brandt's voles genotype I (Figure 1). The Brandt's voles genotype I showed significant differences from other known Cryptosporidium spp. or genotypes in the SSU rRNA sequences. Except for the isolate WY42 is identical with the known sequence (MH187877, C. suis), sequence heterogeneity was observed in other two known Cryptosporidium species/genotypes. The sequences clustered with Cryptosporidium muskrat genotype II exhibit two nucleotide insertions (A at position 461 and T at position 469). Three types of sequences were seen in Cryptosporidium environmental sequences with some substitutions (Table 2).
Figure 1. Phylogenetic analysis of Cryptosporidium spp. using Neighbor-Joining (NJ) method based on sequences of the small subunit ribosomal RNA (SSU rRNA) gene. Bootstrap values >50% are shown (1,000 replicates). Isolates obtained in the present study are indicated by solid square. The SSU rRNA gene sequence of Eimeria tenella is used as the outgroup.
Table 2. Variations in the SSU nucleotide sequences among Cryptosporidium environmental sequences in the present study.
Discussion
Cryptosporidium spp. is one of an important apicomplexan parasite. Many studies have shown that Cryptosporidium spp. can infect humans and animals, and many Cryptosporidium species/genotypes exhibiting public health significance have been found (7). However, it is rarely reported in rodents, especially in voles (19, 20). In this study, we first characterized the prevalence of Cryptosporidium spp. in Brandt's voles.
The prevalence of Cryptosporidium spp. infection varies with species and sampling locations.
In the present study, the overall prevalence of Cryptosporidium spp. in Brandt's voles was 18.7%, which was higher than that in Qinghai voles (8.9%, 8/90) from China (21), common voles (14.2%, 50/353) and bank voles (7.1%, 10/140) from Europe (22), and lower than that in common voles (22.6%, 74/328) from Czech Republic (23), in common voles (73%, 200/274) from Poland (24), and in meadow voles (52.4%; 163/311) from USA (22). The sample size may also be the causation of the difference in prevalence. Furthermore, the prevalence difference between female and male Brandt's voles was not significant, but there were significant differences in body weight and sampling location, respectively. To a certain degree, Rodent's weight can represent its age. The present study showed that prevalence of Cryptosporidium spp. in Brandt's voles was negatively correlated with age, and the youngest voles were significantly higher than the other two groups, which is consistent with previous reports (25). Stronger immunity in older Brandt's voles may lower the infection rates. Both DWQ and MD have similar climatic conditions, while MD is experiencing more anthropogenic disturbances, such as village and grazing activity, compared with DWQ, which may contribute to the difference of parasite prevalence (26).
C. suis, a zoonotic potential species of Cryptosporidium, are commonly detected in pigs (27–29). Other host, such as Cervus unicolor (Reference not published, access number: KX668209), Vulpes vulpes (Reference not published, access number: MN996816), and Apodemus flavicollis (20), were also found to be infected by the parasite. As far as we know, this species was first reported in Brandt's Vole, which suggests that Brandt's Vole might be a potential source of human cryptosporidiosis. Other two known species/genotypes, Cryptosporidium environmental sequence and Cryptosporidium muskrat genotype II, have been found in other environmental samples (30–32), which suggests that environment plays an important role in transmission dynamics of the parasites. Future studies to characterize the prevalence of the parasites in environmental samples from the grassland areas is needed.
Moreover, several loci differences exist in the sequences of Cryptosporidium environmental sequence and Cryptosporidium muskrat genotype II which are in line with previous studies that the heterogeneity of Cryptosporidium SSU sequence was higher (22). Previous studies have shown that the host range of Cryptosporidium genotypes found in arvicolinae is relatively limited (e.g., Cryptosporidium muskrat II were commonly detected in voles than other hosts), which may be the result of host divergence (22). Whether these novel genotype found in this survey is Brandt's vole specific remains to be further studied (23).
Conclusion
In summary, this study first described the prevalence of Cryptosporidium spp. in Brandt's vole worldwide. Four Cryptosporidium species/genotypes, including a known zoonotic species, were identified in the study area, implicating Brandt's vole could be a potential source of human Cryptosporidium infection. Further studies focusing on more host (herdsman, cattle, sheep etc.) as well as source of water to evaluate the transmission network of Cryptosporidium spp., especially zoonotic species, in this pastoral area is needed.
Data Availability Statement
The nucleotide sequences generated in the present study have been deposited in GenBank under accession numbers MT108810 - MT108826.
Ethics Statement
The animal study was reviewed and approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences.
Author Contributions
HH and SF designed the experiments. SF collected the samples. HC, YW, CH, and SH performed the DNA extraction and PCR. SF and HC analyzed the data. SF wrote the manuscript. HH revised the manuscript. All authors contributed to the article and approved the submitted version.
Funding
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDA19050204); State Administration of Forestry and Grassland and Chinese Academy of Sciences (grant number CZBZX-1).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
1. Innes EA, Chalmers RM, Wells B, Pawlowic MC. A one health approach to tackle cryptosporidiosis. Trends Parasitol. (2020) 36:290–303. doi: 10.1016/j.pt.2019.12.016
2. Jenkins MB, Eaglesham BS, Anthony LC, Kachlany SC, Bowman DD, Ghiorse WC. Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum oocysts. Appl Environ Microbiol. (2010) 76:1926–34. doi: 10.1128/AEM.02295-09
3. Chalmers RM, Robinson G, Elwin K, Elson R. Analysis of the Cryptosporidium spp. and gp60 subtypes linked to human outbreaks of cryptosporidiosis in England and Wales, 2009 to 2017. Parasit Vect. (2019) 12:95. doi: 10.1186/s13071-019-3354-6
4. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet. (2013) 382:209–22. doi: 10.1016/S0140-6736(13)60844-2
5. Rabiee MH, Mahmoudi A, Siahsarvie R, Krystufek B, Mostafavi E. Rodent-borne diseases and their public health importance in Iran. PLoS Negl Trop Dis. (2018) 12:e0006256. doi: 10.1371/journal.pntd.0006256
6. Zhang Z, Pech R, Davis S, Shi D, Wan X, Zhong WJO. Extrinsic and intrinsic factors determine the eruptive dynamics of Brandt's voles microtus brandti in inner Mongolia, China. Oikos. (2003) 100:299–310. doi: 10.1034/j.1600-0706.2003.11810.x
7. Feng Y, Ryan UM, Xiao L. Genetic diversity and population structure of Cryptosporidium. Trends Parasitol. (2018) 34:997–1011. doi: 10.1016/j.pt.2018.07.009
8. Feng SY, Chang H, Luo J, Huang JJ, He HX. First report of enterocytozoon bieneusi and Cryptosporidium spp. in peafowl (Pavo cristatus) in China. Int J Parasitol Parasites Wildl. (2019) 9:1–6. doi: 10.1016/j.ijppaw.2019.03.014
9. Xiao X, Qi R, Han HJ, Liu JW, Qin XR, Fang LZ, et al. Molecular identification and phylogenetic analysis of Cryptosporidium, Hepatozoon and Spirometra in snakes from central China. Int J Parasitol Parasites Wildl. (2019) 10:274–80. doi: 10.1016/j.ijppaw.2019.10.001
10. Ryan U. Cryptosporidium in birds, fish and amphibians. Exp Parasitol. (2010) 124:113–20. doi: 10.1016/j.exppara.2009.02.002
11. Certad G, Follet J, Gantois N, Hammouma-Ghelboun O, Guyot K, Benamrouz-Vanneste S, et al. Prevalence, molecular identification, and risk factors for cryptosporidium infection in edible marine fish: a survey across sea areas surrounding France. Front Microbiol. (2019) 10:1037. doi: 10.3389/fmicb.2019.01037
12. Li G, Yin B, Wan X, Wei W, Wang G, Krebs CJ, et al. Successive sheep grazing reduces population density of Brandt's voles in steppe grassland by altering food resources: a large manipulative experiment. Oecologia. (2016) 180:149–59. doi: 10.1007/s00442-015-3455-7
13. Zhang M, He H. Parasite-mediated selection of major histocompatibility complex variability in wild Brandt's voles (Lasiopodomys brandtii) from Inner Mongolia, China. BMC Evol Biol. (2013) 13:149. doi: 10.1186/1471-2148-13-149
14. Nolan MJ, Jex AR, Haydon SR, Stevens MA, Gasser RB. Molecular detection of Cryptosporidium cuniculus in rabbits in Australia. Infect Genet Evol. (2010) 10:1179–87. doi: 10.1016/j.meegid.2010.07.020
15. Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali RJ, et al. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol. (1999) 65:1578–83. doi: 10.1128/AEM.65.4.1578-1583.1999
16. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. (2016) 33:1870–4. doi: 10.1093/molbev/msw054
17. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. (1987) 4:406–25.
18. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. (1985) 39:783–91. doi: 10.1111/j.1558-5646.1985.tb00420.x
19. Khan A, Shaik JS, Grigg ME. Genomics and molecular epidemiology of Cryptosporidium species. Acta Trop. (2018) 184:1–14. doi: 10.1016/j.actatropica.2017.10.023
20. Danisova O, Valencakova A, Stanko M, Luptakova L, Hatalova E, Canady A. Rodents as a reservoir of infection caused by multiple zoonotic species/genotypes of C. parvum, C. hominis, C. suis, C. scrofarum, and the first evidence of C. muskrat genotypes I and II of rodents in Europe. Acta Trop. (2017) 172:29–35. doi: 10.1016/j.actatropica.2017.04.013
21. Zhang X, Jian Y, Li X, Ma L, Karanis G, Karanis P. The first report of Cryptosporidium spp. in Microtus fuscus (Qinghai vole) and Ochotona curzoniae (wild plateau pika) in the Qinghai-Tibetan Plateau area, China. Parasitol Res. (2018) 117:1401–7. doi: 10.1007/s00436-018-5827-5
22. Stenger BLS, Horcickova M, Clark ME, Kvac M, Condlova S, Khan E, et al. Cryptosporidium infecting wild cricetid rodents from the subfamilies Arvicolinae and Neotominae. Parasitology. (2018) 145:326–34. doi: 10.1017/S0031182017001524
23. Horcickova M, Condlova S, Holubova N, Sak B, Kvetonova D, Hlaskova L, et al. Diversity of Cryptosporidium in common voles and description of Cryptosporidium alticolis sp. n. and Cryptosporidium microti sp. n. (Apicomplexa: Cryptosporidiidae). Parasitology. (2019) 146:220–33. doi: 10.1017/S0031182018001142
24. Bajer A, Bednarska M, Pawelczyk A, Behnke JM, Gilbert FS, Sinski E. Prevalence and abundance of Cryptosporidium parvum and Giardia spp. in wild rural rodents from the Mazury Lake District region of Poland. Parasitology. (2002) 125:21–34. doi: 10.1017/S0031182002001865
25. Feng SY, Chang H, Li FH, Wang CM, Luo J, He HX. Prevalence and molecular characterization of Trichomonas gallinae from domestic pigeons in Beijing, China. Infect Genet Evol. (2018) 65:369–72. doi: 10.1016/j.meegid.2018.08.021
26. Young H, Griffin RH, Wood CL, Nunn CL. Does habitat disturbance increase infectious disease risk for primates? Ecol Lett. (2013) 16:656–63. doi: 10.1111/ele.12094
27. Xiao L, Moore JE, Ukoh U, Gatei W, Lowery CJ, Murphy TM, et al. Prevalence and identity of Cryptosporidium spp. in pig slurry. Appl Environ Microbiol. (2006) 72:4461–3. doi: 10.1128/AEM.00370-06
28. Wang H, Zhang Y, Wu Y, Li J, Qi M, Li T, et al. Occurrence, molecular characterization, and assessment of zoonotic risk of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Pigs in Henan, Central China. J Eukaryot Microbiol. (2018) 65:893–901. doi: 10.1111/jeu.12634
29. Petersen HH, Jianmin W, Katakam KK, Mejer H, Thamsborg SM, Dalsgaard A, et al. Cryptosporidium and Giardia in Danish organic pig farms: seasonal and age-related variation in prevalence, infection intensity and species/genotypes. Vet Parasitol. (2015) 214:29–39. doi: 10.1016/j.vetpar.2015.09.020
30. Jiang JL, Alderisio KA, Xiao LH. Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Appl Environ Microbiol. (2005) 8:4446–54. doi: 10.1128/AEM.71.8.4446-4454.2005
31. Ruecker NJ, Matsune JC, Wilkes G, Lapen DR, Topp E, Edge TA, et al. Molecular and phylogenetic approaches for assessing sources of Cryptosporidium contamination in water. Water Res. (2012) 16:5135–50. doi: 10.1016/j.watres.2012.06.045
Keywords: prevalence, public health, zoonoses, phylogenetic analysis, genotyping
Citation: Feng S, Chang H, Wang Y, Huang C, Han S and He H (2020) Molecular Characterization of Cryptosporidium spp. in Brandt's Vole in China. Front. Vet. Sci. 7:300. doi: 10.3389/fvets.2020.00300
Received: 13 March 2020; Accepted: 04 May 2020;
Published: 30 June 2020.
Edited by:
Simona Gabrielli, Sapienza University of Rome, ItalyReviewed by:
Longxian Zhang, Henan Agricultural University, ChinaQuan Liu, Foshan University, China
Copyright © 2020 Feng, Chang, Wang, Huang, Han and He. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Hongxuan He, hehx@ioz.ac.cn