mmu-miR-374b-5p modulated inflammatory factors via downregulation of C/EBP ß/NF-κB signaling in Kupffer cells during Echinococcus multilocularis infection.

BACKGROUND: Alveolar echinococcosis (AE) is an important infectious disease caused by the metacestode larvae of Echinococcus multilocularis, seriously threatening global public health security. Kupffer cells (KCs) play important roles in liver inflammatory response. However, their role in hepatic alveolar echinococcosis has not yet been fully elucidated. METHODS: In this study, qRT-PCR was used to detect the expression level of miR-374b-5p in KCs. The target gene of miR-374b-5p was identified through luciferase reporter assays and loss of function and gains. Critical genes involved in NFκB signaling pathway were analyzed by qRT-PCR and western blot. RESULTS: This study reported that miR-374b-5p was significantly upregulated in KCs during E. multilocularis infection and further showed that miR-374b-5p was able to bind to the 3'-UTR of the C/EBP ß gene and suppressed its expression. The expression levels of NF-κBp65, p-NF-κBp65 and pro-inflammatory factors including iNOS, TNFα and IL6 were attenuated after overexpression of miR-374b-5p while enhanced after suppression of miR-374b-5p. However, the Arg1 expression level was promoted after overexpression of miR-374b-5p while suppressed after downregulation of miR-374b-5p. Additionally, increased protein levels of NF-κBp65 and p-NF-κBp65 were found in the C/EBP ß-overexpressed KCs. CONCLUSIONS: These results demonstrated that miR-374b-5p probably regulated the expression of inflammatory factors via C/EBP ß/NF-κB signaling. This finding is helpful to explore the mechanism of inflammation regulation during E. multilocularis infection.

mmu-miRNA-342-3p promotes hepatic stellate cell activation and hepatic fibrosis induced by Echinococcus multilocularis infection via targeting Zbtb7a.

Liver fibrosis is one of the histopathological characters during Echinococcus multilocularis infection. The activation of hepatic stellate cells (HSCs) is a key event in the development of liver fibrosis. However, the molecular mechanism of HSC activation in the E. multilocularis infection-induced liver fibrosis remains largely unclear. Here, we reported that mmu-miR-342-3p was most dominantly expressed in HSCs and was upregulated in the HSCs in response to E. multilocularis infection. We further showed that mmu-miR-342-3p was able to bind to the 3' UTR of the Zbtb7a gene and regulated its expression. Moreover, mmu-miR-342-3p expression was negatively correlated with its target gene Zbtb7a in HSCs during E. multilocularis infection. Knockdown of mmu-miR-342-3p promoted the expression of Gfap in the activated HSCs in vitro. In the E. multilocularis-infected mice, knockdown of mmu-miR-342-3p suppressed the expression of α-Sma, Col1α1, and TGF-ß but promoted the expression of Gfap. Therefore, mmu-miR-342-3p is a key regulator for activation of HSCs, and inhibiting mmu-miR-342-3p to suppressed Zbtb7a-mediated TGF-ß signaling in activated HSCs could be a novel strategy to treat liver fibrosis induced by E. multilocularis.

Proteomic profiling of serum extracellular vesicles identifies diagnostic markers for echinococcosis.

Echinococcosis is a parasitic disease caused by the metacestodes of Echinococcus spp. The disease has a long latent period and is largely underdiagnosed, partially because of the lack of effective early diagnostic approaches. Using liquid chromatography-mass spectrometry, we profiled the serum-derived extracellular vesicles (EVs) of E. multilocularis-infected mice and identified three parasite-origin proteins, thioredoxin peroxidase 1 (TPx-1), transitional endoplasmic reticulum ATPase (TER ATPase), and 14-3-3, being continuously released by the parasites into the sera during the infection via EVs. Using ELISA, both TPx-1 and TER ATPase were shown to have a good performance in diagnosis of experimental murine echinococcosis as early as 10 days post infection and of human echinococcosis compared with that of control. Moreover, TER ATPase and TPx-1 were further demonstrated to be suitable for evaluation of the prognosis of patients with treatment. The present study discovers the potential of TER ATPase and TPx-1 as promising diagnostic candidates for echinococcosis.

Expansion of Opportunistic Enteric Fungal Pathogens and Occurrence of Gut Inflammation in Human Liver Echinococcosis.

Increasing evidence shows that the gut fungal mycobiota is implicated in human disease. However, its relationship with chronic helminth infections, which cause immunosuppression and affect over 1 billion people worldwide, remains unexplored. In this study, we investigated the gut mycobiome and its associations with gut homeostasis in a severe helminth disease worldwide: liver echinococcosis. Fecal samples from 63 patients and 42 healthy controls were collected to characterize the fungal signatures using ITS1 sequencing, QIIME pipeline, and machine learning analysis. The levels of fecal calprotectin and serological anti-Saccharomyces cerevisiae antibodies (ASCA) in these subjects were experimentally measured. We found that fungal microbiota was significantly skewed in disease, with an overrepresentation of Aspergillus, Candida, Geotrichum, Kazachstania, and Penicillium and a decrease of Fusarium. Machine learning analysis revealed that the altered fungal features could efficiently predict infection with high sensitivity and specificity (area under the curve [AUC] = 0.93). The dysbiosis was characterized by expansions of multiple opportunistic pathogens (Aspergillus spp. and Candida spp.). Clinical association analysis revealed that host immunity might link to the expansions of the invasive fungi. Accompanying the opportunistic pathogen expansion, the levels of fungi-associated fecal calprotectin and serological ASCA in the patients were elevated, suggesting that gut inflammation and microbiota translocation occurred in this generally assumed extraintestinal disease. This study highlights enteric fungal pathogen expansions and increased levels of markers for fungi-associated mucosal inflammation and intestinal permeability as hallmarks of liver echinococcosis. IMPORTANCE Helminth infection affects over 1 billion people worldwide. However, its relationship with the gut mycobiome remains unknown. Among the most prevalent helminth diseases, human hydatid disease (echinococcosis) is highlighted as one of the most important (second/third for alveolar/cystic echinococcosis) foodborne parasitic diseases at the global level. Herein, we investigated the mycobiome and gut homeostasis (i.e., inflammation and permeability) in human echinococcosis. Our results revealed that fungal dysbiosis with an expansion of opportunistic pathogens and increased levels of fecal calprotectin and serum ASCA are hallmarks of human liver echinococcosis. Host immunity is associated with enteric fungal expansions. These findings suggest that an extraintestinal helminth infection is able to alter gut fungal microbiota and impair gut homeostasis, which resembles concomitant gut symptoms in inflammatory gut-related diseases (e.g., AIDS). In clinical practice, physicians need to take cautious medical consideration of gut health for nonintestinal helminth diseases.

HIV protease inhibitor nelfinavir is a potent drug candidate against echinococcosis by targeting Ddi1-like protein.

BACKGROUND: Alveolar echinococcosis (AE), which is caused by larval Echinococcus multilocularis, is one of the world's most dangerous neglected diseases. Currently, no fully effective treatments are available to cure this disease. METHODS: In vitro protoscolicidal assay along with in vivo murine models was applied in repurposing drugs against AE. Genome-wide identification and homology-based modeling were used for predicting drug targets. RNAi, enzyme assay, and RNA-Seq analyses were utilized for investigating the roles in parasite survival and validations for the drug target. FINDINGS: We identified nelfinavir as the most effective HIV protease inhibitor against larval E. multilocularis. Once-daily oral administration of nelfinavir for 28 days resulted in a remarkable reduction in parasite infection in either immune-competent or immunocompromised mice. E. multilocularis DNA damage-inducible 1 protein (EmuDdi1) is predicted as a target candidate for nelfinavir. We proved that EmuDdi1 is essential for parasite survival and protein excretion and acts as a functionally active protease for this helminth. We found nelfinavir is able to inhibit the proteolytic activity of recombinant EmuDdi1 and block the EmuDdi1-related pathways for protein export. With other evidence of drug efficacy comparison, our results suggest that inhibition of EmuDdi1 is a mechanism by which this HIV proteinase inhibitor mediates its antiparasitic action on echinococcosis. INTERPRETATION: This study demonstrates that nelfinavir is a promising candidate for treating echinococcosis. This drug repurposing study proves that the widely prescribed drug for AIDS treatment is potent in combating E. multilocularis infection and thus provides valuable insights into the development of single-drug therapy for highly prevalent co-infection between HIV and helminth diseases. FUNDING: This work was supported by the National Natural Science Foundation of China (31802179), the Natural Science Foundation of Gansu Province, China (No. 21JR7RA027), and the State Key Laboratory of Veterinary Etiological Biology (No. SKLVEB2021YQRC01).

A chromosome-level genome assembly for the rabbit tapeworm Taenia pisiformis.

Taenia pisiformis is one of the most widespread gastrointestinal parasites and its larvae (cysticercosis) causes significant economic loss to rabbit industry. No efficient drug is available for this disease to date. To better understand its genomics, we assembled a 211-Mb high quality genome of T. pisiformis at chromosome level with a scaffold N50 size of 20 Mbp. Totally, 12,097 protein-coding genes was predicted from the genome. Genome-level phylogenetic analysis confirmed the taxonomic affiliations with other tapeworms and revealed that T. pisiformis diverged from its closely related relative T. hydatigena âˆ¼ 14.6 Mya. Comparative genomic analyses revealed that the T. pisiformis genome was characterized by adaptive features of strong positive selection signals from carbohydrate/lipid metabolism and body surface integrity, and of expanded gene families related to metabolism of amino acids and lipids. The high-quality genome of T. pisiformis constitutes a resource for the comparative genomics and for further applications in general parasitology.

Efficient delivery of Echinococcus multilocularis miRNAs using chitosan nanoparticles.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease, a great threat to human health due to limited interventions. microRNAs are a type of small non-coding RNA that plays a key role in many diseases and is considered as a potential therapeutic target for control of parasitic diseases. However, naked miRNAs are difficult to enter into cells and are easily degraded in both external and internal environments. Chitosan (CS) has recently been used as a promising vehicle for delivery of nucleic acids. Therefore, we prepared miRNA-bearing CS nanoparticles and investigated the physicochemical properties as well as the delivery efficiency. We found that CS nanoparticles was relatively stable, offered miRNA strong protection from degradation and had low cytotoxicity with no significant effects on cell proliferation and apoptosis. CS nanoparticles were shown to be easily absorbed by cells and have remarkable liver tropism. Furthermore, CS nanoparticles were used to efficiently deliver E. multilocularis miR-4989 in vitro and in vivo and caused a significant reduction in the expression of UBE2N in the liver, a potential target of emu-miR-4989, at both mRNA and protein levels. Our data demonstrate that CS nanoparticles can act as a vehicle for efficient liver-targeted delivery of miRNAs and for development of miRNA-based therapeutics against E. multilocularis infection.

Integrative Analysis of RNA Expression and Regulatory Networks in Mice Liver Infected by Echinococcus multilocularis.

The larvae of Echinococcus multilocularis causes alveolar echinococcosis, which poses a great threat to the public health. However, the molecular mechanisms underlying the host and parasite interactions are still unclear. Exploring the transcriptomic maps of mRNA, miRNA and lncRNA expressed in the liver in response to E. multilocularis infection will help us to understand its pathogenesis. Using liver perfusion, different cell populations including the hepatic cells, hepatic stellate cells and Kupffer cells were isolated from mice interperitoneally inoculated with protoscoleces. Their transcriptional profiles including lncRNAs, miRNAs and mRNAs were done by RNA-seq. Among these cell populations, the most differentially-expressed (DE) mRNA, lncRNAs and miRNAs were annotated and may involve in the pathological processes, mainly including metabolic disorders, immune responses and liver fibrosis. Following the integrative analysis of 38 differentially-expressed DEmiRNAs and 8 DElncRNAs, the lncRNA-mRNA-miRNA networks were constructed, including F63-miR-223-3p-Fbxw7/ZFP36/map1b, F63-miR-27-5p-Tdrd6/Dip2c/Wdfy4 and IFNgAS1-IFN-γ. These results unveil the presence of several potential lncRNA-mRNA-miRNA axes during E. multilocularis infection, and further exploring of these axes may contribute to better understanding of the pathogenic mechanisms.

circRNA Expression Pattern and circRNA-miRNA-mRNA Network in HCs, HSCs, and KCs of Murine Liver After Echinococcus multilocularis Infection.

Caused by Echinococcus multilocularis (E. multilocularis), alveolar echinococcosis is reported every year around the world and severely threatens the safety of human beings and animals. However, the molecular interaction relationships between host and E. multilocularis still remains unclear. With multiple functions, circRNA plays a crucial role in regulating the development of a parasitic disease. With that in mind, the main purpose of this study was to reveal the circRNA expression profiles and circRNA-miRNA-mRNA network relationships in hepatocytes (HCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) of murine liver after E. multilocularis infection. After sequencing, 6,290 circRNAs were identified from 12 hepatic cell samples. Based on the subsequent analysis, 426 and 372 circRNAs were significantly different in HC expression at 2 and 3 months after E. multilocularis infection, and similar results were also demonstrated in HSCs (426 and 372 circRNAs) and KCs (429 and 331 circRNAs), respectively. Eight candidate circRNAs were randomly selected to identify the accuracy of the sequencing results by using qRT-PCR. Additionally, three circRNAs-miRNA-mRNA networks in HCs, HSCs, and KCs were constructed. Taken together, our study provided a systematic presentation of circRNAs in murine liver cells after E. multilocularis infection, and these networks are essential for research in circRNAs associated with E. multilocularis infection.

Molecular characterization and immune protection of the 3-hydroxyacyl-CoA dehydrogenase gene in Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (CE) is a serious parasitic zoonosis caused by the larvae of the tapeworm Echinococcus granulosus. The development of an effective vaccine is one of the most promising strategies for controlling CE. METHODS: The E. granulosus 3-hydroxyacyl-CoA dehydrogenase (EgHCDH) gene was cloned and expressed in Escherichia coli. The distribution of EgHCDH in protoscoleces (PSCs) and adult worms was analyzed using immunofluorescence. The transcript levels of EgHCDH in PSCs and adult worms were analyzed using quantitative real-time reverse transcription PCR (RT-qPCR). The immune protective effects of the rEgHCDH were evaluated. RESULTS: The 924-bp open reading frame sequence of EgHCDH, which encodes a protein of approximately 34 kDa, was obtained. RT-qPCR analysis revealed that EgHCDH was expressed in both the PSCs and adult worms of E. granulosus. Immunofluorescence analysis showed that EgHCDH was mainly localized in the tegument of PSCs and adult worms. Western blot analysis showed that the recombinant protein was recognized by E. granulosus-infected dog sera. Animal challenge experiments demonstrated that dogs immunized with recombinant (r)EgHCDH had significantly higher serum IgG, interferon gamma and interleukin-4 concentrations than the phosphate-buffered saline (PBS) control group. The rEgHCDH vaccine was able to significantly reduce the number of E. granulosus and inhibit the segmental development of E. granulosus compared to the PBS control group. CONCLUSIONS: The results suggest that rEgHCDH can induce partial immune protection against infection with E. granulosus and could be an effective candidate for the development of new vaccines.

Molecular cloning and functional characterization of a thioredoxin peroxidase gene in Echinococcus multilocularis.

Thioredoxin peroxidase (TPx) plays an important role in protecting parasites against oxidative damage. However, studies on the role of TPxs in Echinococcus multilocularis are limited. In this study, one tpx gene of E. multilocularis, named as emtpx-1, was identified. EmTPx-1 shares two positionally conserved cysteine residues (Cys48 and Cys169) with orthologs from other platyhelminths. EmTPx-1 is highly expressed in the germinal layer and present in exosome-like vesicles secreted by E. multilocularis metacestodes. EmTPx-1 displays peroxidase activity, which removes hydrogen peroxide in the presence of dithiothreitol. Furthermore, EmTPx-1 could protect DNA from oxidative damages, and EmTPx-1-expressing E. coli cells had an enhanced resistance to oxidative stress. In addition, EmTPx-1 enhanced the expression of arg1, ym1, and il-10, but suppressed inos, tnf-α, and il-1ß expression in LPS-stimulated macrophages. Our data suggest a critical role for EmTPx-1 in oxidative stresses and M2 macrophage polarization.

Echinococcus multilocularis infection induces UBE2N suppression via exosomal emu-miR-4989.

Echinococcus multilocularis metacestodes mainly reside in liver in humans and animals, and cause serious damages. UBE2N was herein shown to be downregulated in response to the infection. UBE2N was further shown to be predominantly expressed in the hepatocytes, which was also significantly downregulated during the infection. UBE2N was a target of emu-miR-4989, which was loaded into the exosomes secreted by parasites. These emu-miR-4989-encapsulating exosomes were internalized by hepatocytes, and induced a significant decrease of relative luciferase activity in the cells transfected with the construct containing a wild type of UBE2N 3'-UTR compared to the control (p < 0.05). These results demonstrate that emu-miR-4989 is involved in the UBE2N inhibition in the hepatocytes during E. multilocularis through exosomes.

Comparative Proteomic Analysis of Different Parts of Taenia Hydatigena.

Taenia hydatigena, a globally distributed parasite, is a canine tapeworm and causes huge economic losses in the food industry. Using LC-MS/MS, the proteomes of T. hydatigena cyst scolex, designated as CS, and the cyst without the scolex, designated as CWS, were profiled and a total of 764 different proteins were identified, 664 of which were identified in CS, 412 identified in CWS, and 312 in both. Comparative analysis revealed that CS had more abundant proteins associated with growth and development, while CWS had more abundant proteins constituting a scaffolding and protective extracellular matrix. Consistent with the sequencing data, the abundance of the five selected proteins was validated to be higher in CWS than CS by Western blotting. The current data will provide a clue for further pinpointing a role of these proteins in the biology of T. hydatigena.

Identification of Different Extracellular Vesicles in the Hydatid Fluid of Echinococcus granulosus and Immunomodulatory Effects of 110 K EVs on Sheep PBMCs.

Echinococcosis, mainly caused by Echinococcus granulosus, is one of the 17 neglected tropical diseases. Extracellular vesicles (EVs) play an essential role in the host-parasite interplay. However, the EVs in the hydatid fluid (HF) of E. granulosus are not fully characterized. Herein, three different types of HF EVs, designated as 2 K, 10 K, and 110 K EVs based on the centrifugal force used, were morphologically identified. A total of 97, 80, and 581 proteins were identified in 2 K, 10 K, and 110 K EVs, respectively, 39 of which were commonly shared. Moreover, 11, 8, and 25 miRNAs were detected, respectively, and all of the 7 selected miRNAs were validated by qPCR to be significantly lower abundant than that in protoscoleces. It was further deemed that 110 K EVs were internalized by sheep peripheral blood mononuclear cells (PBMCs) in a time-dependent manner and thus induced interleukin (IL)-10, tumor necrosis factor-α (TNF-α), and IRF5 were significantly upregulated and IL-1ß, IL-17, and CD14 were significantly downregulated (p < 0.05). These data demonstrate the physical discrepancy of three HF EVs and an immunomodulatory effect of 110 K EVs on sheep PMBCs, suggesting a role in immune responses during E. granulosus infection.

Characterization of Fructose-1,6-Bisphosphate Aldolase 1 of Echinococcus multilocularis.

Glycolysis is one of the important ways by which Echinococcus multilocularis acquires energy. Fructose-1, 6-bisphosphate aldolase (FBA) plays an important role in this process, but it is not fully characterized in E. multilocularis yet. The results of genome-wide analysis showed that the Echinococcus species contained four fba genes (FBA1-4), all of which had the domain of FBA I and multiple conserved active sites. EmFBA1 was mainly located in the germinal layer and the posterior of the protoscolex. The enzyme activity of EmFBA1 was 67.42 U/mg with Km and Vmax of 1.75 mM and 0.5 mmol/min, respectively. EmFBA1 was only susceptible to Fe3+ but not to the other four ions (Na+, Ca2+, K+, Mg2+), and its enzyme activity was remarkably lost in the presence of 0.5 mM Fe3+. The current study reveals the biochemical characters of EmFBA1 and is informative for further investigation of its role in the glycolysis in E. multilocularis.

Proteomics Analysis of Hydatigera taeniaeformis Metacestode Stage.

Hydatigera taeniaeformis (H. taeniaeformis) is one of the most robust of tapeworm parasites that is widely distributed around the world. Information of proteins of H. taeniaeformis has not previously been reported. Using liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, the proteome of H. taeniaeformis metacestode was profiled and a total of 408 proteins were identified. Of these, 26.5% (108/408) were annotated to be associated with metabolic pathways. Consistently, Gene Ontology analysis showed that those identified proteins were mainly classified into metabolic process of the biological processes. A set of metabolic enzymes, including Fructose-1,6-bisphosphate aldolase, enolase, Glucan phosphorylase, and phosphoenolpyruvate carboxykinase, were abundant in H. taeniaeformis metacestodes. In addition, some rare but interesting proteins like antigens (such as tegument protein and Antigen B) were identified. The two recombinant proteins of tegument protein and Antigen B were well-recognized by the sera from the H. taeniaeformis-infected mice. The H. taeniaeformis metacestode proteome might help to find new candidates for the immunodiagnosis and vaccine development and provide valuable information on H. taeniaeformis biology.

Profiling of miRNAs in Mouse Peritoneal Macrophages Responding to Echinococcus multilocularis Infection.

Alveolar echinococcosis (AE) is a zoonotic helminthic disease caused by infection with the larval of Echinococcus multilocularis in human and animals. Here, we compared miRNA profiles of the peritoneal macrophages of E. multilocularis-infected and un-infected female BALB/c mice using high-throughput sequencing. A total of 87 known miRNAs were differentially expressed (fold change ≥ 2, p < 0.05) in peritoneal macrophages in mice 30- and 90-day post infection compared with ones in un-infected mice. An increase of mmu-miR-155-5p expression was observed in peritoneal macrophages in E. multilocularis-infected mice. Compared with the control group, the production of nitric oxide (NO) was increased in peritoneal macrophages transfected with mmu-miR-155-5p mimics at 12 h after transfection (p < 0.001). Two key genes (CD14 and NF-κB) in the LPS/TLR4 signaling pathway were also markedly altered in mmu-miR-155-5p mimics transfected cells (p < 0.05). Moreover, mmu-miR-155-5p mimics suppressed IL6 mRNA expression and promoted IL12a and IL12b mRNA expression. Luciferase assays showed that mmu-miR-155-5p was able to bind to the 3' UTR of the IKBKE gene and decreased luciferase activity. Finally, we found the expression of IKBKE was significantly downregulated in both macrophages transfected with mmu-miR-155-5p and macrophages isolated from E. multilocularis-infected mice. These results demonstrate an immunoregulatory effect of mmu-miR-155 on macrophages, suggesting a role in regulation of host immune responses against E. multilocularis infection.

miRNA-seq of Echinococcus multilocularis Extracellular Vesicles and Immunomodulatory Effects of miR-4989.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease. In the infected mice, emu-miR-4989-3p is present in sera, but its role remains unknown. Using high-throughput sequencing and qPCR, emu-miR-4989-3p was herein confirmed to be encapsulated into E. multilocularis extracellular vesicles. In the transfected macrophages, emu-miR-4989-3p was demonstrated to significantly inhibit NO production compared to the control (p < 0.05). Moreover, transfection of emu-miR-4989-3p also gave rise to the increased expression of TNF-α (p < 0.01). Furthermore, emu-miR-4989-3p induced the dysregulation of several key components in the LPS/TLR4 signaling pathway compared with the control, especially TLR4 and NF-κB that both were upregulated. Conversely, the NO production and the expression of TNF-α, TLR4 and NF-κB tended to be increased and decreased in the mimics-transfected cells upon emu-miR-4989-3p low expression, respectively. These results suggest that emu-miR-4989-3p is one of 'virulence' factors encapsulated into the extracellular vesicles, potentially playing a role in the pathogenesis of E. multilocularis.

Profiling circulating microRNAs in serum of Fasciola gigantica-infected buffalo.

Circulating miRNAs are stably existed in serum and plasma and can serve as a novel class of biomarkers for the diagnosis of helminthic infection. Fasciola gigantica, the causative agents of fascioliasis, live in the liver of in humans and ruminants, especially cattle, goat and sheep. In this study, a total of 121 host circulating miRNAs were differentially expressed (2 ≥ fold change, p < 0.05), of which 44 miRNAs were up-regulated and 77 miRNAs were significantly down-regulated. Consistent with the sequencing data, qRT-PCR results showed that the expression levels of bta-miR-21-5p and bta-miR-23a were elevated gradually and bta-miR-125a was decreased gradually at the F. gigantica infection time points. Four F. gigantica-specific miRNAs, including three known miRNAs (fgi-miR-87, fgi-miR-71, and fgi-miR-124), and one novel miRNA (novel miR-1) were identified in the sera of F. gigantica-infected buffaloes. Further analyses demonstrated that two parasite-derived miRNAs (fgi-miR-87 and fgi-miR-71) were specifically detected in sera of F. gigantica-infected buffaloes. These findings will be helpful to understand the roles of circulating miRNAs in host-parasite interaction and to potentiate serum miRNAs as diagnostic targets for F. gigantica.

High-throughput identification of microRNAs in Taenia hydatigena, a cestode threatening livestock breeding industry.

Infection of Cysticercus tenuicollis, the larval stage of Taenia hydatigena, is extensively found in sheep and pigs and jeopardizes the breeding and meat industry. miRNAs are a subclass of small noncoding regulatory RNAs and closely associated with the pathogenesis and biology of parasites. Here, using HiSeq sequencing we identified 49 known and 2 potential novel miRNAs in C. tenuicollis, of which both thy-miR-71 and -87 were predominant. Using RT-qPCR, 6 selected miRNAs were validated, and thy-miR-71 and -miR-87 were confirmed to be highly expressed, with the copy number of approximately 82,340 ±â€¯2079 and 19,580 ±â€¯609 per 1 ng total RNA, respectively. Similar to other cestodes, T. hydatigena was predicted to have two conserved miRNA clusters thy-miR-71/2c/2b and thy-miR-4989/277, and three members of the former were confirmed to reside sequentially within the genomic region of 253 bp by PCR. The current data provide us a valuable resource for further studies of a role of miRNAs in T. hydatigena biology and infection.