Interaction between tissue-dwelling helminth and the gut microbiota drives mucosal immunoregulation.

Tissue-dwelling helminths affect billions of people around the world. They are potent manipulators of the host immune system, prominently by promoting regulatory T cells (Tregs) and are generally associated with a modified host gut microbiome. However, the role of the gut microbiota in the immunomodulatory processes for these non-intestinal parasites is still unclear. In the present study, we used an extra-intestinal cestode helminth model-larval Echinococcus multilocularis to explore the tripartite partnership (host-helminth-bacteria) in the context of regulating colonic Tregs in Balb/c mice. We showed that larval E. multilocularis infection in the peritoneal cavity attenuated colitis in Balb/c mice and induced a significant expansion of colonic Foxp3+ Treg populations. Fecal microbiota depletion and transplantation experiments showed that the gut microbiota contributed to increasing Tregs after the helminth infection. Shotgun metagenomic and metabolic analyses revealed that the gut microbiome structure after infection was significantly shifted with a remarkable increase of Lactobacillus reuteri and that the microbial metabolic capability was reprogrammed to produce more Treg cell regulator-short-chain fatty acids in feces. Furthermore, we also prove that the L. reuteri strain elevated in infected mice was sufficient to promote the colonic Treg frequency and its growth was potentially associated with T cell-dependent immunity in larval E. multilocularis infection. Collectively, these findings indicate that the extraintestinal helminth drives expansions of host colonic Tregs through the gut microbes. This study suggests that the gut microbiome serves as a critical component of anti-inflammation effects even for a therapy based on an extraintestinal helminth.

Modulation of long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression in the liver of Beagle dogs by Toxocara canis infection.

BACKGROUND: Long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) play crucial roles in regulating various physiological and pathological processes. However, the role of lncRNAs and mRNAs in mediating the liver response during Toxocara canis infection remains incompletely understood. METHODS: In the present study, the expression profile of lncRNAs and mRNAs was investigated in the liver of Beagle dogs infected by T. canis using high-throughput RNA sequencing. RESULTS: Compared with the control groups, 876 differentially expressed (DE) lncRNAs and 288 DEmRNAs were identified at 12 h post-infection (hpi), 906 DElncRNAs and 261 DEmRNAs were identified at 24 hpi, and 876 DElncRNAs and 302 DEmRNAs were identified at 36 days post-infection (dpi). A total of 16 DEmRNAs (e.g. dpp4, crp and gnas) were commonly identified at the three infection stages. Enrichment and co-localization analyses identified several pathways involved in immune and inflammatory responses during T. canis infection. Some novel DElncRNAs, such as LNC_015756, LNC_011050 and LNC_011052, were also associated with immune and inflammatory responses. Also, LNC_005105 and LNC_005401 were associated with the secretion of anti-inflammatory cytokines, which may play a role in the healing of liver pathology at the late stage of infection. CONCLUSIONS: Our data provided new insight into the regulatory roles of lncRNAs and mRNAs in the pathogenesis of T. canis and improved our understanding of the contribution of lncRNAs and mRNAs to the immune and inflammatory response of the liver during T. canis infection.

Identification and expression analysis of a new small ubiquitin-like modifier from Taenia pisiformis.

The small ubiquitin-like modifier (SUMO) plays important roles, with the SUMOylation pathway as one of its core components. In the present work, a single SUMO gene was initially identified from Taenia pisiformis and designated as TpSUMO. Bioinformatic analysis showed that the TpSUMO gene contained a 309 bp open reading frame (ORF), encoding 102 amino acids, and had a predicted molecular weight of ∼12 kDa. The amino acid sequence of TpSUMO was deduced and it shared 44.00% identity with human SUMO2 (HsSUMO2) and exhibited more than 97.78% identity with SUMOs from Taenia and Echinococcus. TpSUMO possessed a putative non-consensus site (FK11MG) within its N-terminus and a typical di-glycine (GG) motif at the C-terminus. Basic local alignment search tool (BLAST) analysis showed that only a single SUMO-related ortholog was present in each set of known genome data for fourteen tapeworm species. The precursor His-TpSUMO-FL, mature His-TpSUMO-GG and mutant His-TpSUMO-GGK11R proteins (∼18 kDa) were expressed in Escherichia coli Rosseta (DE3), and rabbit polyclonal anti-TpSUMO was generated with a high titer of 1.28 × 105. In vitro SUMOylation assay results showed that TpSUMO multimer formation in the His-TpSUMO-GG reaction could be catalyzed by the human SAE1/SAE2 and UBC9 conjugation system, but K11R mutation disrupted TpSUMO chain synthesis. Quantitative real-time PCR (qRT-PCR) further revealed that TpSUMO was ubiquitously expressed in different stages of T. pisiformis and in higher levels during an early development phase (day 14) of adult worms. Immunofluorescence localization showed that TpSUMO was detected in the bladder wall of cysticerci, in the testis in immature segment, and within eggs in the gravid proglottids. These findings indicated that TpSUMO is a new member of the SUMO protein family and may play a vital role in regulation of functions within proteins involved in worm growth and development.

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.

Echinococcosis Is Associated with the Increased Prevalence of Intestinal Blastocystis Infection in Tibetans and Host Susceptibility to the Blastocystis in Mice.

Blastocystis is a common human intestinal protozoan parasite. Little is known about its prevalence in echinococcosis. This study tested whether Echinococcus multilocularis infection would increase host susceptibility to Blastocystis. A total of 114 fecal samples (68 hydatid disease patients and 46 healthy people) were collected from Tibetans in the Qinghai province in China. The presence of Blastocystis was identified by sequencing of the small subunit (SSU) rRNA gene. Balb/c mice were co-infected with Blastocystis and E. multilocularis and tested for host susceptibility to Blastocystis. The overall Blastocystis prevalence was 12.3%; 16.2% in the patients and 4.4% in healthy people (p < 0.05). Sequence analysis identified three known Blastocystis genotypes, including ST1, ST2, and ST3, and one unknown genotype. Experimental dual infection significantly reduced mouse survival rate (20%), induced more severe signs, and increased intestinal damages with a higher intestinal colonization level of Blastocystis. The mouse model showed that E. multilocularis infection increases host susceptibility to Blastocystis. Our study shows a significantly higher prevalence of Blastocystis in patients with liver echinococcosis and reveals that non-intestinal E. multilocularis infection increases host susceptibility to the Blastocystis. Our results highlight that E. multilocularis infection is associated with Blastocystis. These findings remind us that more attention should be paid to the gut health of the patients with a helminth infection during clinical patient care.

Extracellular vesicles from Fasciola gigantica induce cellular response to stress of host cells.

Extracellular vesicles (EVs) from parasitic helminths play an important role in immunomodulation. However, EVs are little studied in the important parasite Fasciola gigantica. Here the ability of EVs from F. gigantica to induce cellular response to stress (reactive oxygen species generation, autophage and DNA damage response) in human intrahepatic biliary epithelial cells (HIBEC) was investigated. F. gigantica-derived EVs were isolated by ultracentrifugation, and identified with transmission electron microscopy, nanoparticle size analysis and parasite-derived EV markers. Internalization of EVs by HIBEC was determined by confocal immunofluorescence microscopy and flow cytometry. ROS levels in HIBEC were detected by molecular probing. EVs-induced autophagy and DNA-damaging effects were determined by evaluating expression levels of light chain 3B protein (LC3B), phosphor- H2A.X and phosphor-Chk1, respectively. Results revealed that EVs with sizes predominately ranging from 39 to 110 nm in diameter were abundant in adult F. gigantica and contained the parasite-derived marker proteins enolase and 14-3-3, and EVs were internalized by HIBEC. Further, uptake of EVs into HIBEC was associated with increased levels of reactive oxygen species, LC3Ⅱ, phosphor-H2A.X and phosphor-Chk1, suggesting EVs are likely to induce autophagy and DNA damage & repair processes. These results indicate F. gigantica EVs are associated with modulations of host cell responses and have a potential important role in the host-parasite interactions.

The genome of the thin-necked bladder worm Taenia hydatigena reveals evolutionary strategies for helminth survival.

Taenia hydatigena is a widespread gastrointestinal helminth that causes significant health problems in livestock industry. This parasite can survive in a remarkably wide range of intermediate hosts and affects the transmission dynamics of zoonotic parasites. T. hydatigena is therefore of particular interest to researchers interested in studying zoonotic diseases and the evolutionary strategies of parasites. Herein we report a high-quality draft genome for this tapeworm, characterized by some hallmarks (e.g., expanded genome size, wide integrations of viral-like sequences and extensive alternative splicing during development), and specialized adaptations related to its parasitic fitness (e.g., adaptive evolutions for teguments and lipid metabolism). Importantly, in contrast with the evolutionarily close trematodes, which achieve gene diversification associated with immunosuppression by gene family expansions, in T. hydatigena and other cestodes, this is accomplished by alternative splicing and gene loss. This indicates that these two classes have evolved different mechanisms for survival. In addition, molecular targets for diagnosis and intervention were identified to facilitate the development of control interventions. Overall, this work uncovers new strategies by which helminths evolved to interact with their hosts.

Newly excysted juveniles of Fasciola gigantica trigger the release of water buffalo neutrophil extracellular traps in vitro.

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes and are among the first line of immune system defense. PMNs can form neutrophil extracellular traps (NETs) in response to some pathogens. The release of NETs plays an important role in trapping and killing invading parasites. However, the effects of NETs on parasitic trematode infections remain unclear. In the present study, water buffalo NET formation, triggered by the newly excysted juveniles (NEJs) of Fasciola gigantica, was visualized by scanning electron microscopy. The major components of the structure of NETs were characterized by immunofluorescence. Viability of flukes incubated with water buffalo PMNs were examined under light microscopy. The results revealed that F. gigantic juveniles triggered PMN-mediated NETs. These NETs were confirmed to comprise the classic characteristics of NETs: DNA, histones, myeloperoxidase and neutrophil elastase. Although NETs were formed in response to viable larvae, the larvae were not killed in vitro. These results suggest that NET formation may serve as a mechanism to hamper the migration of large larvae to facilitate immune cells to kill them. This study demonstrates, for the first time, that parasitic trematode juveniles can trigger NET formation.

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.

Mitochondrial genome of Paruterina candelabraria (Cestoda: Paruterinidae), with implications for the relationships between the genera Cladotaenia and Paruterina.

The taxonomic concept for the family Paruterinidae is controversial, especially concerning the position of the genus Cladotaenia, since the latter genus has been placed sometimes in other families, i.e. in the Taeniidae or in the distinct family Cladotaeniidae; finding a solution based on morphological data is difficult and molecular data on paruterinids and related groups are scarce. In this study, the complete mitochondrial (mt) genome sequence of the type-species of the type-genus of the Paruterinidae, Paruterina candelabraria, was determined and annotated. Gene arrangements are identical with those of Cladotaenia vulturi but differing from those of species of the family Taeniidae by the order change between tRNA-SerUCN and tRNA-LeuCUN. Phylogenetic tree was constructed by Bayesian Inference (BI) analysis using the concatenated amino acid sequences of 12 protein-coding genes. The analysis clearly shows that the Paruterinidae and Taeniidae are sister-groups, and Cladotaenia is a sister taxon of Paruterina. This supports the position of the genus Cladotaenia in the family Paruterinidae and reveals the necessity for sequencing additional taxa of the Paruterinidae for better understanding of phylogenetic relationships within the group.

Molecular cloning and characterization of leucine aminopeptidase gene from Taenia pisiformis.

Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention.

Expression profiles of genes involved in TLRs and NLRs signaling pathways of water buffaloes infected with Fasciola gigantica.

Infection of ruminants and humans with Fasciola gigantica is attracting increasing attention due to its economic impact and public health significance. However, little is known of innate immune responses during F. gigantica infection. Here, we investigated the expression profiles of genes involved in Toll-like receptors (TLRs) and NOD-like receptors (NLRs) signaling pathways in buffaloes infected with 500F. gigantica metacercariae. Serum, liver and peripheral blood mononuclear cell (PBMC) samples were collected from infected and control buffaloes at 3, 10, 28, and 70days post infection (dpi). Then, the levels of 12 cytokines in serum samples were evaluated by ELISA. Also, the levels of expression of 42 genes, related to TLRs and NLRs signaling, in liver and PBMCs were determined using custom RT2 Profiler PCR Arrays. At 3 dpi, modest activation of TLR4 and TLR8 and the adaptor protein (TICAM1) was detected. At 10 dpi, NF-κB1 and Interferon Regulatory Factor signaling pathways were upregulated along with activation of TLR1, TLR2, TLR6, TLR10, TRAF6, IRF3, TBK1, CASP1, CD80, and IFNA1 in the liver, and inflammatory response with activated TLR4, TLR9, TICAM1, NF-κB1, NLRP3, CD86, IL-1B, IL-6, and IL-8 in PBMCs. At 28 dpi, there was increase in the levels of cytokines along with induction of NLRP1 and NLRP3 inflammasomes-dependent immune responses in the liver and PBMCs. At 70 dpi, F. gigantica activated TLRs and NLRs, and their downstream interacting molecules. The activation of TLR7/9 signaling (perhaps due to increased B-cell maturation and activation) and upregulation of NLRP3 gene were also detected. These findings indicate that F. gigantica alters the expression of TLRs and NLRs genes to evade host immune defenses. Elucidation of the roles of the downstream effectors interacting with these genes may aid in the development of new interventions to control disease caused by F. gigantica infection.

Serum levels of cytokines in water buffaloes experimentally infected with Fasciola gigantica.

Fasciola gigantica infection in water buffaloes causes significant economic losses especially in developing countries. Although modulation of the host immune response by cytokine neutralization or vaccination is a promising approach to control infection with this parasite, our understanding of cytokine's dynamic during F. gigantica infection is limited. To address this, we quantified the levels of serum cytokines produced in water buffaloes following experimental infection with F. gigantica. Five buffaloes were infected via oral gavage with 500 viable F. gigantica metacercariae and blood samples were collected from buffaloes one week before infection and for 13 consecutive weeks thereafter. The levels of 10 cytokines in serum samples were simultaneously determined using ELISA. F. gigantica failed to elicit the production of various pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), IL-2, IL-6, IL-12, and IFN-γ. On the other hand, evidence of a Th2 type response was detected, but only early in the course of parasite colonization and included modest increase in the levels of IL-10 and IL-13. The results also revealed suppression of the immune responses as a feature of chronic F. gigantica infection in buffaloes. Taken together, F. gigantica seems to elicit a modest Th2 response at early stage of infection in order to downregulate harmful Th1- and Th17-type inflammatory responses in experimentally infected buffaloes. The full extent of anti-F. gigantica immune response and its relation to pathogenesis requires further study.

TsPKA-r: a potential immunodiagnostic antigen for the detection of porcine cysticercosis.

Cysticercosis, caused by metacestodes of Taenia solium, has a significant soci-economic impact and is of considerable importance in public health. However, there are no specific diagnostic antigens to distinguish between T. solim and Taenia hydatigena. In the present study, cAMP-dependent protein kinase regulatory subunit (TsPKA-r), an excretory/secretary (ES) antigen of T. solium, was used to establish a specific and sensitive diagnostic tool for detection of porcine cysticercosis. The full-length sequence encoding TsPKA-r was amplified by PCR, sequenced and then identified by bioinformatics. The fusion protein with 6×His-tags was expressed in E. coli, purified by Ni Sepharose™ 6 Fast Flow and used to test reactionogenicity by immunoblotting. TsPKA-r based indirect enzyme-linked immunosorbent assays (iELISA) showed good performance in recognition of sera of pigs experimentally infected with T. solium metacestodes, with 93.88% sensitivity and 96.40% specificity. There were no cross-reactions against the sera from pigs experimentally infected with T. hydatigena, Toxoplasma gondii or Trichinella spiralis. These results indicate that the TsPKA-r is a promising immunodiagnostic antigen for detection of porcine cysticercosis.

Regulatory effects of Echinococcus multilocularis extracellular vesicles on RAW264.7 macrophages.

Extracellular vesicles (EVs) play a role in intercellular communications via exchanging biological molecules, being involved in host-parasite interplay. Little is to date known about E. multilocularis EVs and their biological activities. Here spherical EVs secreted by E. multilocularis metacestodes were shown to range predominately from 34nm to 95nm in diameter. A total of 433 proteins were identified in the EVs, and the proteins involved in binding (42%) and catalytic activity (41%) were most frequently represented. Moreover, the proteins associated with EV biogenesis and trafficking, including annexin, 14-3-3, tetraspanin and heat shock protein 70kDa, were highly enriched. It was shown that the EVs remarkably suppressed NO produced by activated RAW macrophages via downregulation of inducible nitric oxide synthase expression (p <0.01). Suppression of pro-inflammatory cytokines, especially IL-1α and IL-1ß, was also observed post treatment with the EVs. Conversely, increased expression of the majority (10/11) of key components involved in the LPS/TLR4 pathway was induced by the EVs. These results demonstrate a regulatory effect of E. multilocularis EVs on macrophages, suggesting a role in parasite-host interactions.

Transcriptomic responses of water buffalo liver to infection with the digenetic fluke Fasciola gigantica.

BACKGROUND: Fasciola gigantica, the tropical liver fluke, infects buffaloes in Asian and African countries and causes significant economic losses and poses public health threat in these countries. However, little is known of the transcriptional response of buffaloes to infection with F. gigantica. The objective of the present study was to perform the first transcriptomic analysis of buffalo liver infected by F. gigantica. Understanding the mechanisms that underpin F. gigantica infection in buffaloes will contribute to our ability to control this parasite. METHODS: We challenged buffaloes with 500 viable F. gigantica metacercariae and collected liver samples through a time course at 3, 42 and 70 days post-infection (dpi). Then, we performed gene expression analysis on liver samples using RNA sequencing (RNA-Seq) Illumina technology and confirmed the RNA-Seq data by quantitative RT-PCR analysis. RESULTS: Totals of 496, 880 and 441 differentially expressed transcripts were identified in the infected livers at 3, 42 and 70 dpi, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that transcriptional changes in the liver of infected buffaloes evolve over the course of infection. The predominant response of buffaloes to infection was mediated by certain pathways, such as MHC antigen processing and presentation, Toll-like receptor 4 (TLR4), transforming growth factor beta (TGF-ß), and the cytochrome P450. Hepatic drug metabolizing enzymes and bile secretion were also affected. CONCLUSIONS: Fasciola gigantica can induce statistically significant and biologically plausible differences in the hepatic gene expression of infected buffaloes. These findings provide new insights into the response of buffaloes to F. gigantica over the course of infection, which may be useful in determining pathways that can modulate host-parasite interaction and thus potentially important for clearance of the parasite.

Moniezia benedeni and Moniezia expansa are distinct cestode species based on complete mitochondrial genomes.

Moniezia spp. parasitize the intestines of ruminants, causing monieziasis. In this study, the complete mitochondrial (mt) genomes of M. benedeni and M. expansa have been determined, characterized and employed to test the hypothesis that M. benedeni and M. expansa are distinct species by phylogenetic analysis based on the concatenated amino acid sequences derived from 12 protein-coding genes, inferred with Bayesian and Maximum-likelihood methods. The complete mt genomes of M. benedeni and M. expansa were 13,958bp and 13,934bp in size, respectively. Nucleotide sequence identity between the two mt genomes was 83.4%. Each of the two circular mt genomes encodes 36 genes including two ribosomal RNA genes, 22 transfer RNA genes and 12 protein-coding genes, which are transcribed from the same direction. The gene orders of the two mt genomes are identical to those of Anoplocephala spp. (Anoplocephalidae), Hymenolepis spp. (Hymenolepididae) and Dipylidium caninum (Dipylidiidae), but distinct from the species of the Taeniidae family. Phylogenetic analysis confirmed that M. benedeni and M. expansa are taxonomically valid species and have a sister relationship, regardless of the analytical method employed. Furthermore, comparing the cox1 gene sequences of Moniezia spp. from the NCBI deposited sequences and the ones obtained in the present study revealed that the nucleotide sequence differences were 12.5% for M. benedeni and 6.2% for M. expansa, respectively, suggesting the existence of cryptic species in these parasites. The complete mt genome sequences reported herein will be valuable in further studies of diagnoses, molecular ecology and population genetics of Moniezia spp. of socio-economic importance.