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

Prevalência e caracterização molecular de Cryptosporidium spp. no cervo de Père David (Elaphurus davidianus) em Jiangsu, China

Si-Yang Huang Yi-Min Fan Yi Yang Yi-Jun Ren Jing-Zhi Gong Na Yao Bin Yang About the authors

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 area.

Keywords:
Cryptosporidium; Père David’s deer; 60 kDa glycoprotein (gp60) gene; the small subunit ribosomal RNA (SSU rRNA)

Resumo

Cryptosporidium é um parasita zoonótico que causa diarreia em uma ampla gama de animais, incluindo veados. Pouco se sabe sobre a prevalência e o genótipo de Cryptosporidium spp. no cervo de Père David. Neste estudo, 137 amostras fecais do cervo de Père David foram coletadas entre julho de 2017 e agosto de 2018, na Reserva Dafeng, e analisadas para Cryptosporidium spp. por nested-PCR baseado no gene do RNA ribossômico da subunidade pequena (SSU rRNA), seguido de análises de sequências para determinar as espécies. O gene da glicoproteína de 60 kDa (gp60) foi utilizado para caracterizar Cryptosporidium spp. Dentre as 137 amostras, 2 (1,46%) foram positivas para Cryptosporidium spp. de acordo com os resultados do sequenciamento gênico de SSU rRNA. Ambas as amostras pertenciam ao genótipo do cervo Cryptosporidium, com duas deleções nucleotídicas e uma substituição nucleotídica. Os dados de prevalência e a caracterização molecular deste estudo fornecem conhecimentos básicos para controlar e prevenir infecções por Cryptosporidium nos cervos de Père David nessa.

Palavras-chave:
Cryptosporidium; cervos de Père David; Gene de glicoproteína de 60 kDa (gp60); RNA ribossômico da pequena subunidade (SSU rRNA)

Introduction

Cryptosporidiosis is caused by Cryptosporidium spp., which is an important enteric apicomplexan parasite of zoonosis in the world (Parsons et al., 2015Parsons MB, Travis D, Lonsdorf EV, Lipende I, Roellig DMA, Kamenya S, et al. Epidemiology and molecular characterization of Cryptosporidium spp. in humans, wild primates, and domesticated animals in the Greater Gombe Ecosystem, Tanzania. PLoS Negl Trop Dis 2015; 9(2): e0003529. http://dx.doi.org/10.1371/journal.pntd.0003529. PMid:25700265.
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; Tanriverdi et al., 2007Tanriverdi S, Blain JC, Deng B, Ferdig MT, Widmer G. Genetic crosses in the apicomplexan parasite Cryptosporidium parvum define recombination parameters. Mol Microbiol 2007; 63(5): 1432-1439. http://dx.doi.org/10.1111/j.1365-2958.2007.05594.x. PMid:17302818.
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). It is a critical emerging infectious disease in humans and animals that can lead to diarrhea or other serious symptoms (Zhao et al., 2015Zhao GH, Du SZ, Wang HB, Hu XF, Deng MJ, Yu SK, et al. First report of zoonotic Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi in golden takins (Budorcas taxicolor bedfordi). Infect Genet Evol 2015; 34: 394-401. http://dx.doi.org/10.1016/j.meegid.2015.07.016. PMid:26190449.
http://dx.doi.org/10.1016/j.meegid.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, 1999Deng MQ, Cliver DO. Improved immunofluorescence assay for detection of Giardia and Cryptosporidium from asymptomatic adult cervine animals. Parasitol Res 1999; 85(8-9): 733-736. http://dx.doi.org/10.1007/s004360050623. PMid:10431741.
http://dx.doi.org/10.1007/s004360050623...
).

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., 2018Ding Y, Ding J, Li P, Zhu J. Strategy study of wild Pere David deer (Elaphurus davidianus) population development in China. J Jiangsu Forestry Sci Technol 2018; 45: 49-51.).

Currently, more than 30 Cryptosporidium species and genotypes have been identified (Baroudi et al., 2018Baroudi D, Hakem A, Adamu H, Amer S, Khelef D, Adjou K, et al. Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria. Parasit Vectors 2018; 11(1): 582. http://dx.doi.org/10.1186/s13071-018-3172-2. PMid:30400983.
http://dx.doi.org/10.1186/s13071-018-317...
; Ryan et al., 2014Ryan U, Fayer R, Xiao L. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 2014; 141(13): 1667-1685. http://dx.doi.org/10.1017/S0031182014001085. PMid:25111501.
http://dx.doi.org/10.1017/S0031182014001...
). Eleven of them, C. muris, C. parvum, Cryptosporidium muskrat II genotype, C. hominis-like genotype, Cryptosporidium caribou genotype, C. hominis, C. bovis, C. ryanae, Cryptosporidium deer genotype, C. ubiquitum and Cryptosporidium suis-like genotype, have been identified in cervids, including red deer, sika deer, white-tailed deer, roe deer, caribou and moose in China, Czech Republic, Japan, the United Kingdom, Spain, the United States, Norway and Poland (Garcia-Presedo et al., 2013García-Presedo I, Pedraza-Díaz S, González-Warleta M, Mezo M, Gómez-Bautista M, Ortega-Mora LM, et al. The first report of Cryptosporidium bovis, C. ryanae and Giardia duodenalis sub-assemblage A-II in roe deer (Capreolus capreolus) in Spain. Vet Parasitol 2013; 197(3-4): 658-664. http://dx.doi.org/10.1016/j.vetpar.2013.07.002. PMid:23890824.
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; Huang et al., 2018Huang J, Zhang Z, Zhang Y, Yang Y, Zhao J, Wang R, et al. Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in deer in Henan and Jilin, China. Parasit Vectors 2018; 11(1): 239. http://dx.doi.org/10.1186/s13071-018-2813-9. PMid:29650036.
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; Jellison et al., 2009Jellison KL, Lynch AE, Ziemann JM. Source tracking identifies deer and geese as vectors of human-infectious Cryptosporidium genotypes in an urban/suburban watershed. Environ Sci Technol 2009; 43(12): 4267-4272. http://dx.doi.org/10.1021/es900081m. PMid:19603633.
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; Kato et al., 2016Kato S, Yanagawa Y, Matsuyama R, Suzuki M, Sugimoto C. Molecular identification of the Cryptosporidium deer genotype in the Hokkaido sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. Parasitol Res 2016; 115(4): 1463-1471. http://dx.doi.org/10.1007/s00436-015-4880-6. PMid:26687968.
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; Kotkova et al., 2016Kotková M, Nemejc K, Sak B, Hanzal V, Kvetonova D, Hlaskova L, et al. Cryptosporidium ubiquitum, C. muris and Cryptosporidium deer genotype in wild cervids and caprines in the Czech Republic. Folia Parasitol (Praha) 2016; 63: 003. https://doi.org/10.14411/fp.2016.003.
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; Siefker et al., 2002Siefker C, Rickard LG, Pharr GT, Simmons JS, O’Hara TM. Molecular characterization of Cryptosporidium sp. isolated from northern Alaskan caribou (Rangifer tarandus). J Parasitol 2002; 88(1): 213-216. http://dx.doi.org/10.1645/0022-3395(2002)088[0213:MCOCSI]2.0.CO;2. PMid:12053974.
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; Wang et al., 2008Wang R, Wang J, Sun M, Dang H, Feng Y, Ning C, et al. Molecular characterization of the Cryptosporidium cervine genotype from a sika deer (Cervus nippon Temminck) in Zhengzhou, China and literature review. Parasitol Res 2008; 103(4): 865-869. http://dx.doi.org/10.1007/s00436-008-1069-2. PMid:18575889.
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; Wells et al., 2015Wells B, Shaw H, Hotchkiss E, Gilray J, Ayton R, Green J, et al. Prevalence, species identification and genotyping Cryptosporidium from livestock and deer in a catchment in the Cairngorms with a history of a contaminated public water supply. Parasit Vectors 2015; 8(1): 66. http://dx.doi.org/10.1186/s13071-015-0684-x. PMid:25650114.
http://dx.doi.org/10.1186/s13071-015-068...
). Little information is available about Cryptosporidium infections in Père David’s deer (Huang et al., 2018Huang J, Zhang Z, Zhang Y, Yang Y, Zhao J, Wang R, et al. Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in deer in Henan and Jilin, China. Parasit Vectors 2018; 11(1): 239. http://dx.doi.org/10.1186/s13071-018-2813-9. PMid:29650036.
http://dx.doi.org/10.1186/s13071-018-281...
). In this study, the prevalence of Cryptosporidium infections and molecular characteristics were investigated in Père David’s deer in the Dafeng Reserve, China.

Materials and Methods

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., 2013Zhao GH, Ren WX, Gao M, Bian QQ, Hu B, Cong MM, et al. Genotyping Cryptosporidium andersoni in cattle in Shaanxi Province, Northwestern China. PLoS One 2013; 8(4): e60112. http://dx.doi.org/10.1371/journal.pone.0060112. PMid:23560072.
http://dx.doi.org/10.1371/journal.pone.0...
). 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., 2003Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 2003; 41(6): 2744-2747. http://dx.doi.org/10.1128/JCM.41.6.2744-2747.2003. PMid:12791920.
http://dx.doi.org/10.1128/JCM.41.6.2744-...
; Feng et al., 2012Feng Y, Karna SR, Dearen TK, Singh DK, Adhikari LN, Shrestha A, et al. Common occurrence of a unique Cryptosporidium ryanae variant in zebu cattle and water buffaloes in the buffer zone of the Chitwan National Park, Nepal. Vet Parasitol 2012; 185(2-4): 309-314. https://doi.org/10.1016/j.vetpar.2011.09.025.
https://doi.org/10.1016/j.vetpar.2011.09...
; Li et al., 2014Li N, Xiao LH, Alderisio K, Elwin K, Cebelinski E, Chalmers R, et al. Subtyping Cryptosporidium ubiquitum, a Zoonotic Pathogen Emerging in Humans. Emerg Infect Dis 2014; 20(2): 217-224. http://dx.doi.org/10.3201/eid2002.121797. PMid:24447504.
http://dx.doi.org/10.3201/eid2002.121797...
). 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.

Table 1
Primers used in the study, annealing temperatures used in the PCR, and expected sizes of the PCR products.

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.

Results and Discussion

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., 2016Kotková M, Nemejc K, Sak B, Hanzal V, Kvetonova D, Hlaskova L, et al. Cryptosporidium ubiquitum, C. muris and Cryptosporidium deer genotype in wild cervids and caprines in the Czech Republic. Folia Parasitol (Praha) 2016; 63: 003. https://doi.org/10.14411/fp.2016.003.
https://doi.org/10.14411/fp.2016.003...
), 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., 2018Huang J, Zhang Z, Zhang Y, Yang Y, Zhao J, Wang R, et al. Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in deer in Henan and Jilin, China. Parasit Vectors 2018; 11(1): 239. http://dx.doi.org/10.1186/s13071-018-2813-9. PMid:29650036.
http://dx.doi.org/10.1186/s13071-018-281...
; Kato et al., 2016Kato S, Yanagawa Y, Matsuyama R, Suzuki M, Sugimoto C. Molecular identification of the Cryptosporidium deer genotype in the Hokkaido sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. Parasitol Res 2016; 115(4): 1463-1471. http://dx.doi.org/10.1007/s00436-015-4880-6. PMid:26687968.
http://dx.doi.org/10.1007/s00436-015-488...
; Santin & Fayer, 2015Santin M, Fayer R. Enterocytozoon bieneusi, Giardia, and Cryptosporidium infecting white-tailed deer. J Eukaryot Microbiol 2015; 62(1): 34-43. http://dx.doi.org/10.1111/jeu.12155. PMid:25066778.
http://dx.doi.org/10.1111/jeu.12155...
). 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., 2014Huang JY, Yue DY, Qi M, Wang RJ, Zhao JF, Li JQ, et al. Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Ningxia, northwestern China. BMC Vet Res 2014; 10(1): 292. http://dx.doi.org/10.1186/s12917-014-0292-6. PMid:25488627.
http://dx.doi.org/10.1186/s12917-014-029...
).

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. TheCryptosporidium 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., 2018Huang J, Zhang Z, Zhang Y, Yang Y, Zhao J, Wang R, et al. Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in deer in Henan and Jilin, China. Parasit Vectors 2018; 11(1): 239. http://dx.doi.org/10.1186/s13071-018-2813-9. PMid:29650036.
http://dx.doi.org/10.1186/s13071-018-281...
) 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., 2007bFeng Y, Ortega Y, He G, Das P, Xu M, Zhang X, et al. Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Vet Parasitol 2007b; 144(1-2): 1-9. http://dx.doi.org/10.1016/j.vetpar.2006.10.001. PMid:17097231.
http://dx.doi.org/10.1016/j.vetpar.2006....
). 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., 2008Cinque K, Stevens MA, Haydon SR, Jex AR, Gasser RB, Campbell BE. Investigating public health impacts of deer in a protected drinking water supply watershed. Water Sci Technol 2008; 58(1): 127-132. http://dx.doi.org/10.2166/wst.2008.632. PMid:18653946.
http://dx.doi.org/10.2166/wst.2008.632...
; Feng et al., 2007aFeng Y, Alderisio KA, Yang W, Blancero LA, Kuhne WG, Nadareski CA, et al. Cryptosporidium genotypes in wildlife from a New York watershed. Appl Environ Microbiol 2007a; 73(20): 6475-6483. http://dx.doi.org/10.1128/AEM.01034-07. PMid:17720824.
http://dx.doi.org/10.1128/AEM.01034-07...
; Koehler et al., 2016Koehler AV, Haydon SR, Jex AR, Gasser RB. Cryptosporidium and Giardia taxa in faecal samples from animals in catchments supplying the city of Melbourne with drinking water (2011 to 2015). Parasit Vectors 2016; 9:315. https://dx.doi.org/10.1186%2Fs13071-016-1607-1.
https://dx.doi.org/10.1186%2Fs13071-016-...
; Perz & Le Blancq, 2001Perz JF, Le Blancq SM. Cryptosporidium parvum infection involving novel genotypes in wildlife from lower New York State. Appl Environ Microbiol 2001; 67(3): 1154-1162. http://dx.doi.org/10.1128/AEM.67.3.1154-1162.2001. PMid:11229905.
http://dx.doi.org/10.1128/AEM.67.3.1154-...
; Robinson et al., 2011Robinson G, Chalmers RM, Stapleton C, Palmer SR, Watkins J, Francis C, et al. A whole water catchment approach to investigating the origin and distribution of Cryptosporidium species. J Appl Microbiol 2011; 111(3): 717-730. http://dx.doi.org/10.1111/j.1365-2672.2011.05068.x. PMid:21649804.
http://dx.doi.org/10.1111/j.1365-2672.20...
; Xiao et al., 2002Xiao L, Sulaiman IM, Ryan UM, Zhou L, Atwill ER, Tischler ML, et al. Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health. Int J Parasitol 2002; 32(14): 1773-1785. http://dx.doi.org/10.1016/S0020-7519(02)00197-2. PMid:12464424.
http://dx.doi.org/10.1016/S0020-7519(02)...
). 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 is similar to the Cryptosporidium deer genotype reported before; however, compared with the Cryptosporidium deer genotype (GenBank accession numbers: KX259129), there were three mutants. More information and future studies are needed to determine whether this genotype represents a new genotype.

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.

Figure 1
Phylogenetic relationship between SSU rRNA sequences of Cryptosporidium was analyzed using the Kimura-2 parametric model of Neighbor-Joining (NJ). The numbers on branches was the percentage of the bootstrap values in 1000 replicates. The Cryptosporidium isolates identified in this study are represented by black triangles.

Acknowledgements

Project support was provided by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(Veterinary Medicine);

  • How to cite: Huang SY, Fan YM, Yang Y, Ren YJ, Gong JZ, Yao N, et al. Prevalence and molecular characterization of Cryptosporidium spp. in Père David’s deer (Elaphurus davidianus) in Jiangsu, China. Braz J Vet Parasitol 2020;29(2):e017919. https://doi.org/10.1590/S1984-29612020013

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Publication Dates

  • Publication in this collection
    18 May 2020
  • Date of issue
    2020

History

  • Received
    15 Oct 2019
  • Accepted
    16 Dec 2019
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br