Open Access
Volume 23, 2016
Article Number 23
Number of page(s) 8
Published online 27 May 2016

© S. Jiang et al., published by EDP Sciences, 2016

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Cystic echinococcosis (CE) is a chronic parasitic zoonosis caused by infection with the larval stage of the cestode Echinococcus granulosus, resulting in the development of cysts in human and domestic animals. In domestic animals, especially in sheep, which appear to be highly susceptible to infection, CE causes considerable health problems and thereby significantly affects the income of herdsmen.

It has been demonstrated that the pathogenesis of CE is strongly influenced by genetics, and certain MHC polymorphisms are associated with individual susceptibility in humans [1, 2]. In domestic animals, we have carried out several studies to investigate the relationship between MHC-DRB1/DQB1 gene polymorphism and genetic resistance or susceptibility to CE in Chinese Merino, Duolang, and Kazakh sheep [9, 16, 22, 23]. Surprisingly, we have identified genetic markers (MHC MvaIbc-SacIIab-Hin1Iab haplotype) for CE resistance in Kazakh sheep. The rate of E. granulosus infection in the internal organs of Kazakh sheep with this haplotype is confirmed to be significantly lower than in sheep without this haplotype, when exposed to the same number of parasites [22]. CE-resistant sheep therefore have an increased genetic capability to respond to and subsequently reject parasites when challenged.

Subsequently, we made several attempts to understand the mechanism of resistance to CE in sheep with this resistance haplotype. Li et al. found that Th1 cytokines were predominant in sheep with the CE resistance haplotype, which could induce protective immunity, while in sheep without this haplotype, Th2 cytokines were predominant, which could induce susceptibility to CE infection [23]. Hui et al. [17] found that the differences in CE resistance in sheep with different MHC haplotypes could be partially mediated by Th1-type cytokines and IgE, in the early stage of E. granulosus infection. It is therefore concluded that the intestinal tissue of sheep may have a certain function in parasite invasion. It has been demonstrated in mice that intestinal mucosal immunity, as a first line of defense, could be involved in stopping larval growth of E. granulosus at the very first stages [25]. Furthermore, using suppression subtractive hybridization (SSH) methods to detect different expressed sequence tags (ESTs) in the intestinal tissue of sheep infected with E. granulosus, Du et al. [9] found that other immune parameters such as IgM and non-immune factors are involved in induction of these enhanced levels of resistance. However, there remain many unanswered questions about the precise molecular and cellular mechanisms underlying the difference in CE resistance in sheep.

MicroRNAs (miRNAs) are small 20–22 nucleotide (nt) non-coding RNAs, which play a variety of roles in diverse biological processes at the post-transcriptional regulatory level in tissues during animal development [11]. Protozoan miRNAs and components of their silencing machinery possess features different from other eukaryotes, which can provide some clues on the evolution of the RNA-induced silencing machinery. miRNA functions possibly associate with neoblast biology, development, physiology, infection, and immunity of parasites. So far, a number of miRNAs have been identified in parasites, including Plasmodium [7], Echinococcus [3, 8, 24], schistosomula [14, 27], fluke Fasciolopsis buski [5], etc.

Since parasite infection can alter host miRNA expression, it can favor both parasite clearance and infection. miRNA pathways are, thus, a potential target for the therapeutic control of parasitic diseases. However, there is little literature on microRNA profiles in the intestinal tissue after echinococcosis infection, which is the first organ that the parasite invades. It is, therefore, necessary to conduct genome-wide characterization of microRNA profiles involved in resistance to CE infection in sheep, especially in sheep with different MHC haplotypes. In this study, to identify microRNA controlling resistance to CE in the early stage of infection, microRNA profiling was conducted in the intestinal tissue of sheep with resistant (R) and non-resistant (NR) MHC haplotypes after peroral infection with E. granulosus eggs.

Materials and methods

Animals and experimental design

The Animal Care and Use Committee of Shihezi University approved all procedures and experiments. Healthy 2-year-old ewes with CE-resistant [MHCMvaIbc-SacIIab-Hin1Iab(R)] and CE-non-resistant [MHCMvaIbc-SacIIab-Hin1Iab(NR)] haplotypes were selected, according to our previous method [21], and were raised in the farm of Shihezi University. All of the animals were raised under the same conditions of free access to water and food in natural lighting.

These animals were divided into two groups according to their MHC haplotype: three sheep with the CE-resistant (R) haplotype were referred to as group R, while the other three sheep with the CE-non-resistant (NR) haplotype constituted group NR. Sheep were screened for absence of hydatid cysts and were healthy prior to the experiment. According to the previous approach, the sheep were orally infected with E. granulosus eggs [18]. All the animals were sacrificed at 8 hours post-infection. Immediately afterwards, the intestinal tissue was removed and approximately 100 mm3-sized intestinal tissue blocks were collected and frozen immediately in liquid nitrogen prior to long-term storage at −80 °C until RNA extraction.

Small-RNA library preparation and Illumina sequencing

The intestinal tissue was removed from the liquid nitrogen and the total RNA was extracted using TRIZOL (Invitrogen, USA), according to the manufacturer’s protocol. The quality and quantity of RNA samples were checked by an Agilent 2100 Bioanalyzer. The RNA from three sheep in each group was pooled to generate the two libraries. RNA length ranging from 18 to 30 nt was isolated and purified. Proprietary (Solexa) adaptors were then added to the 3′ and 5′-termini of sRNAs and subsequently used for cDNA synthesis. These ligation products were amplified by reverse transcription PCR using an RT-PCR kit (Takara, China). The produced libraries were sequenced using a Solexa sequencer at the Beijing Genomics Institute (BGI)-Shenzhen, China, according to the manufacturer’s instructions. Image analysis and base calling were performed with the Illumina built-in SCS2.8/RTA1.8 software.

Read mapping and annotation

To obtain clean and unique small-RNA reads, the fastx_toolkit (v0.0.13.2, downloadable at was used to filter off low-quality contamination reads. The clean reads were screened against and mapped to the ovine genome assembly (Oar_v3.1, released September 20, 2012) (, using the SOAP (Supplemental Offer and Acceptance Program). Afterwards, we used the BLAST tool on the clean reads against the Rfam database and GenBank to annotate and remove reads for mRNA, tRNA, rRNA, snoRNA, and snRNA. Then, the remaining sequences were searched against the mature miRNAs of animals in miRBase to identify known conserved miRNA homologs in sheep. If small-RNA mature and precursor sequences were perfectly matched to known ovine miRNAs in the miRBase, they were considered conserved miRNAs. Sequences that were identical to or related to the reference mature miRNAs were annotated as miRNA candidates.

Identification of conserved and novel miRNAs

In order to discover potential new miRNAs, the surrounding 300 bases (150 upstream and downstream) flanking each unique miRNA candidate sequence were obtained and their folding secondary structures were determined using the miReap program.

After prediction, the resulting potential miRNA loci were examined carefully based on the distribution and numbers of small RNAs on the entire precursor regions. Those sequences residing in the stem region of the stem-loop structure and ranging between 20 and 22 nt with free energy hybridization lower than −20 kcal/mol were considered as potential new miRNAs.

Determination and target prediction of differentially expressed miRNAs

A list of the differentially expressed genes with a 2-fold difference was generated and controlled with a false discovery rate (FDR) at 0.05 in an experiment. To predict potential miRNA target genes, the differentially expressed miRNAs were analyzed using the MIREAP and TargetScan software to predict potential.

Statistical analysis

All of the data are presented as the means ± SD. A Student’s t-test was performed and P < 0.05 was considered statistically significant, when group comparisons were made.


Summary of the sequence reads

In this study, miRNA from the intestinal tissue of CE-R and CE-NR sheep was sequenced. Firstly, we generated a small-RNA library from sheep with different haplotypes. Illumina sequencing of the purified cDNA libraries generated 28,842,662 total raw reads: 13,800,608 (13.8 million) from the CE-R library and 15,042,054 (15.0 million) from the CE-NR library (Table 1). To assess the efficiency of sequence quality, all reads were annotated and classified by aligning against the miRBase 20.0 database, Rfam database, Repbase database, ovine genome database, and the mRNA database. Of these reads, 72.85% (5.80/7.78 million reads, R) and 76.13% (2.11/4.19 million reads, NR) were mapped to the ovine genome Oar_v3.1. Figure 1 illustrates the summary of data cleaning and length distribution of tags in the two groups. Table 2 displays the information on distribution of small RNA among different groups.

thumbnail Figure 1.

The size distribution of the small RNAs in CE-R and CE-NR libraries. (CE-R: sheep with CE-resistant (R) haplotype; CE-NR: sheep with CE-non-resistant (NR) haplotype).

Table 1.

Summary of reads from raw data to cleaned sequences for small RNAs in CE-R versus CE-NR in sheep.

Table 2.

Summary of known miRNAs in each group.

Identification of known sheep miRNAs and prediction of potential novel miRNAs

To identify known miRNAs in sheep intestines, the data set was compared with the known mammalian miRNAs (miRNA precursors and mature miRNAs) in miRBase 20.0 ( Sequences with a perfect match or one mismatch were retained in the alignment. We identified 351 known miRNAs in the CE-R library and 353 in the CE-NR library (Table 2).

In the present study, we identified three miRNA groups: (a) miRNAs previously described in sheep, (b) miRNAs described in species other than sheep, and (c) new miRNAs without any known annotations. In total, we identified 186 novel miRNAs in the R library and 129 in the NR library.

Differential expression of miRNAs in the CE-R and CE-NR groups

Pairwise comparisons revealed significant differential expression of miRNAs between the two groups. Although some of their expression quantities were equivalent, some miRNAs were expressed differently. We focused on miRNAs meeting our designated criteria of P-values < 0.05 and |log2 (fold change)| > 1, and a total of 83 (75 up and 8 down) differentially expressed miRNAs were selected from the CE-R and CE-NR libraries (Supplementary Table). The normalized count data are given in Supplementary Table, showing the statistically significant differentially expressed miRNAs.

Target gene predictions for differentially expressed miRNAs

A total of 22876 annotated mRNA transcripts were predicted as putative target genes for the 83 differentially expressed miRNAs.


As technological advancements have resulted in increased discovery of new miRNAs, in the present study, using RNA deep sequencing technology, we found a large number of new miRNAs in studied Kazakh sheep. These new miRNAs were all confirmed as having a hairpin secondary structure in their predicted precursor sequences.

The alterations of host miRNA expression reflect the roles of host-derived miRNAs in parasite infection [20]. It is estimated that up to 30% of genes might be regulated by miRNAs. In a separate study, we demonstrated predominant up-regulation of immune factors in the early stage of infection of CE-resistance sheep. However, the regulatory mechanisms for up-regulation were not fully elucidated. The current study has identified 83 differentially expressed miRNAs in the two CE infection sheep groups (resistance and non-resistance), of which, 75 were up-regulated and 8 were down-regulated in the resistance group. The target prediction and integration of these differentially expressed miRNAs with mRNA-seq data generated in our separate study helped to identify the most significant miRNAs and the potential target genes that could be responsible for the resistance ability to CE infection.

Among the known differential expression of miRNAs, miR-21-3p, miR-542-5p, miR-671, miR-134-5p, and miR-26b, miR-27a showed a much higher expression in CE-R sheep compared to the CE-NR library, in terms of normalized read counts (fold changes were 3.37, 3.5, 6.88, 5.25, 3.75, and 3.2, respectively). These miRNAs were already known to be crucially involved in immunity to disease in humans.

In particular, microRNA-21 (miR-21) was found to be up-regulated in cervical cancer tissues [13] Accumulating evidence for differential expression of miR-21 in cervical cancer suggests that it might play a crucial role in tumor biology [15]. It has been confirmed that miR-21 was transactivated through promoter binding of NFKB-P65. In parasite disease, it had been found that inhibition of miR-21 resulted in increased Cryptosporidium parvum burden [28]. Conversely, negative feedback regulation of TLR4/NF-kB signaling may be reached by miR-21 induction after C. parvum infection, as miR-21 targets PDCD4, a proinflammatory protein that promotes activation of NF-kB and suppresses interleukin 10. In the present study, significant overexpression of miR-21 was obtained in CE-resistant sheep, which suggested that it might be a biomarker associated with CE resistance.

Moreover, we also found that microRNA-542-5p was significantly up-regulated in CE-resistant sheep, when compared with their counterparts with non-resistance. MicroRNA-542-5p has been proven to be a novel tumor suppressor in neuroblastoma; overexpression of microRNA-542-5p decreases the invasive potential of neuroblastoma cell lines in vitro [19]. In our study, we find that microRNA-542-5P is highly expressed in sheep with CE resistance, suggesting that it may be related to defense against the parasite.

MiR-134-5p is related to the functioning of B cells. It has been reported that miR-134-5p expression is significantly up-regulated in infectious mononucleosis caused by primary Epstein-Barr virus infection in children [12]. MicroRNA-26b modulates the NF-κB pathway in alveolar macrophages by regulating PTEN [26].

MiR-27a is a member of the miR-27 family and is highly expressed in endothelial cells, where they are involved in angiogenesis, and are also present in the central nervous system, controlling apoptosis [6, 21]. In the present study, miR-27a is up-regulated in the intestinal tissue of sheep with MHC-CE resistance infection with E. granulosus, when compared with sheep without this haplotype, suggesting that miR-27a might play an important role in anti-E. granulosus infection. Up-regulation of the miR-27 family is also observed in Apicomplexan parasite infection; miR-27a is induced in ECM [10] and miR-27b is associated with TLR4-mediated epithelial antimicrobial defense [29] and apoptosis [4].

It is concluded that these microRNAs, related to the inflammation process, especially the NF-kB pathway, may play an important role in fighting E. granulosus infection. However, further studies need to be carried out to verify its function.

Supplementary Table

Supplementary Table

Differential expression levels of miRNAs between CE-R and CE-NR libraries


This study was funded by the National Natural Science Foundation of China (Grant Nos. 31260535 and 31402048) and the Dean Outstanding Youth Fund of the Anhui Academy of Agriculture Sciences (Grant No. 14B0403).


  1. Al-Ghoury AB, El-Hamshary EM, Azazy AA, Hussein EM, Rayan HZ. 2010. HLA class II alleles: susceptibility or resistance to cystic echinococcosis in Yemeni patients. Parasitology Research, 107, 355–361. [CrossRef] [PubMed] [Google Scholar]
  2. Azab ME, Bishara SA, Ramzy RM, Oteifa NM, El-Hoseiny LM, Ahmed MA. 2004. The evaluation of HLA-DRB1 antigens as susceptibility markers for unilocular cystic echinococcosis in Egyptian patients. Parasitology Research, 92, 473–477. [CrossRef] [PubMed] [Google Scholar]
  3. Bai Y, Zhang Z, Jin L, Kang H, Zhu Y, Zhang L, Li X, Ma F, Zhao L, Shi B, Li J, McManus DP, Zhang W, Wang S. 2014. Genome-wide sequencing of small RNAs reveals a tissue-specific loss of conserved microRNA families in Echinococcus granulosus. BMC Genomics, 15, 736. [CrossRef] [PubMed] [Google Scholar]
  4. Cai Y, Chen H, Jin L, You Y, Shen J. 2013. STAT3-dependent transactivation of miRNA genes following Toxoplasma gondii infection in macrophage. Parasite and Vectors, 6, 356. [CrossRef] [Google Scholar]
  5. Chen MX, Hu W, Li J, He JJ, Ai L, Chen JX. 2016. Identification and characterization of microRNAs in the zoonotic fluke Fasciolopsis buski. Parasitology Research, 28, 1–6. [Google Scholar]
  6. Chen Q, Xu J, Li L, Li H, Mao S, Zhang F. 2014. MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis. Cell Death Disease, 5, e1132. [CrossRef] [Google Scholar]
  7. Cohen A, Combes V, Grau GE. 2015. MicroRNAs and Malaria – a dynamic Interaction still incompletely understood. Journal of Neuroinfection Disease, 6(1), 165. [Google Scholar]
  8. Cucher M, Prada L, Mourglia-Ettlin G, Dematteis S, Camicia F, Asurmendi S, Rosenzvit M. 2011. Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes. International Journal for Parasitology, 41(3–4), 439–448. [CrossRef] [PubMed] [Google Scholar]
  9. Du X, Hui W, Zhao B, Guo Y, Ma J, Ma S, Muyesaer B, Liu X, Jia B. 2012. Construction of subtracted library of small intestine in Kazakh sheep with two MHC-DRB1 genotype infected by Echinococcus oncosphere. Journal of Shihezi University (Natural Science), 30(5), 587–591. [Google Scholar]
  10. El-Assaad F, Hempel C, Combes V, Mitchell AJ, Ball HJ, Kurtzhals JA. 2011. Differential microRNA expression in experimental cerebral noncerebral malaria. Infection and Immunology, 79, 2379–2384. [CrossRef] [Google Scholar]
  11. Filipowicz W, Bhattacharyya SN, Sonenberg N. 2008. Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nature Reviews Genetics, 9, 102–114. [CrossRef] [PubMed] [Google Scholar]
  12. Gao L, Ai J, Xie Z, Zhou C, Liu C, Zhang H, Shen K. 2015. Dynamic expression of viral and cellular microRNAs in infectious mononucleosis caused by primary Epstein-Barr virus infection in children. Journal of Virology, 12, 208. [CrossRef] [Google Scholar]
  13. Gocze KGK, Kovacs K, Juhasz K, Gocze P, Kiss I. 2015. MicroRNA Expressions in HPV-induced Cervical Dysplasia and Cancer. Anticancer Research, 35, 523–530. [PubMed] [Google Scholar]
  14. Han H, Peng J, Hong Y, Fu Z, Lu K, Li H, Zhu C, Zhao Q, Lin J. 2015. Comparative analysis of microRNA in schistosomula isolated from non-permissive host and susceptible host. Molecular and Biochemical Parasitology, 204(2), 81–88. [CrossRef] [PubMed] [Google Scholar]
  15. Han Y, Xu GX, Lu H, Yu DH, Ren Y, Wang L, Huang XH, Hou WJ, Wei ZH, Chen YP, Cao YG, Zhang R. 2015. Dysregulation of miRNA-21 and their potential as biomarkers for the diagnosis of cervical cancer. International Journal of Clinical and Experimental Pathology, 8(6), 7131–7139. [PubMed] [Google Scholar]
  16. Hui W, Shen H, Jiang S, Jia B. 2012. MHC-DQB1 variation and its association with resistance or susceptibility to cystic echinococcosis in Chinese Merino sheep. Asian-Austral Journal of Animal Sciences, 12, 1660–1666. [CrossRef] [Google Scholar]
  17. Hui W, Batur M, Du X, Ma S, Ma J, Jia B. 2013. Early antibody, cytokine and chemokine responses during Echinococcus granulosus infection in Kazakh sheep with cystic echinococcosis resistance haplotype. Pakistan Veterinary Journal, 33(2), 205–208. [Google Scholar]
  18. Hui W, Jiang S, Tang J, Hou H, Chen S, Jia B, Ban Q. 2015. An immediate innate immune response occurred in the early stage of E. granulosus eggs infection in sheep: evidence from microarray analysis. PLoS One, 10(8), e0135096. [CrossRef] [PubMed] [Google Scholar]
  19. Bray I, Tivnan A, Bryan K, Foley NH, Watters KM, Tracey L, Davidoff AM, Stallings RL. 2011. MicroRNA-542-5p as a novel tumor suppressor in neuroblastoma. Cancer Letters, 303(1), 56–64. [CrossRef] [PubMed] [Google Scholar]
  20. Jennewein C, Von Knethen A, Schmid T, Brüne B. 2010. MicroRNA-27b contributes to lipopolysaccharide-mediated peroxisome proliferator-activated receptor γ (PPARγ) mRNA destabilization. Journal of Biology Chemistry, 285, 11846–11853. [CrossRef] [Google Scholar]
  21. Judice CC, Bourgard C, Kayano AC, Albrecht L, Costa FT.2016.MicroRNAs in the host-apicomplexan parasites interactions: a review of immunopathological aspects. Frontiers in Cellular and Infection Microbiology, 6, 5. [CrossRef] [PubMed] [Google Scholar]
  22. Li RY, Hui WQ, Jia B, Shi GQ, Zhao ZS, Shen H, Peng Q, Lv LM, Zhou QW, Li HT. 2010. Analysis of the relationship between MHC-DRB1 gene polymorphism and hydatidosis in Kazakh sheep. Asian-Austral Journal of Animal Science, 23, 1145–1151. [CrossRef] [Google Scholar]
  23. Li RY, Hui WQ, Jia B, Shi GQ, Zhao ZS, Shen H, Peng Q, Lv LM, Zhou QW, Li HT. 2011. The relationship between MHC-DRB1 gene second exon polymorphism and hydatidosis resistance of Chinese merino (Sinkiang Junken type), Kazakh and Duolang sheep. Parasite, 18(2), 163–169. [CrossRef] [EDP Sciences] [PubMed] [Google Scholar]
  24. Macchiaroli N, Cucher M, Zarowiecki M, Maldonado L, Kamenetzky L, Rosenzvit MC. 2015. MicroRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach. Parasite and Vectors, 8, 83. [CrossRef] [Google Scholar]
  25. Pater C, Müller V, Harraga M, Liance M, Godot V, Cabbillet JP, Meillet D, Römig T, Vuitton DA. 1998. Intestinal and systemic humoral immunological events in the susceptible Balb/C mouse strain after oral administration of Echinococcus multilocularis eggs. Parasite Immunology, 20, 623–629. [CrossRef] [PubMed] [Google Scholar]
  26. Zhang L, Huang C, Guo Y, Gou X, Hinsdale M, Lloyd P, Liu L. 2015. MicroRNA-26b modulates the NF-κB Pathway in alveolar macrophages by regulating PTEN. Journal of Immunology, 195(11), 5404–5414. [CrossRef] [Google Scholar]
  27. Zhao J, Luo R, Xu X, Zou Y, Zhang Q, Pan W. 2015. High-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation (HITS-CLIP) reveals Argonaute-associated microRNAs and targets in Schistosoma japonicum. Parasites and Vectors, 8, 589. [CrossRef] [Google Scholar]
  28. Zhou R, Hu G, Liu J, Gong AY, Drescher KM, Chen XM. 2009. NF-kappaB p65-dependent transactivation of miRNA genes following Cryptosporidium parvum infection stimulates epithelial cell immune responses. PLoS Pathogens, 5(12), e1000681. [CrossRef] [PubMed] [Google Scholar]
  29. Zhou R, Gong AY, Eischeid AN, Chen XM. 2012. MiR-27b targets KSRP to coordinate TLR4-mediated epithelial defense against Cryptosporidium parvum infection. PLoS Pathogens, 8, e1002702. [CrossRef] [PubMed] [Google Scholar]

Cite this article as: Jiang S, Li X, Wang X, Ban Q, Hui W & Jia B: MicroRNA profiling of the intestinal tissue of Kazakh sheep after experimental Echinococcus granulosus infection, using a high-throughput approach. Parasite, 2016, 23, 23.

All Tables

Table 1.

Summary of reads from raw data to cleaned sequences for small RNAs in CE-R versus CE-NR in sheep.

Table 2.

Summary of known miRNAs in each group.

Supplementary Table

Differential expression levels of miRNAs between CE-R and CE-NR libraries

All Figures

thumbnail Figure 1.

The size distribution of the small RNAs in CE-R and CE-NR libraries. (CE-R: sheep with CE-resistant (R) haplotype; CE-NR: sheep with CE-non-resistant (NR) haplotype).

In the text

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