Improved multiplex PCR assay for host species identification from fecal samples in Echinococcus multilocularis surveillance.

Infection with Echinococcus multilocularis causes alveolar echinococcosis (AE), a severe parasitic disease characterised by progressive hepatic infiltration and fibrosis. Current treatment options remain limited, and the immune-mediated mechanisms underlying fibrosis are not fully understood. This study investigated the role of the IL-33/ST2 axis in AE-associated immunopathology and evaluated the effects of ST2 blockade in an experimental model. A murine model of AE was established in C57BL/6 mice, which were allocated to untreated infected, anti-ST2-treated infected and uninfected control groups. Liver pathology and fibrosis were assessed using haematoxylin and eosin staining, Masson's trichrome staining and transmission electron microscopy. Immune and fibrotic markers were analysed by immunohistochemistry and immunofluorescence. In parallel, liver samples from patients with AE were examined to evaluate translational relevance. Systemic cytokine profiles were quantified to assess immune modulation. Analysis of human AE liver samples showed an increased expression of IL-33/ST2 and associated immune and fibrotic markers, consistent with findings in the experimental mouse model. In infected mice, ST2 blockade significantly reduced metacestode lesion size and hepatic tissue invasion. This was accompanied by reduced fibrosis, as indicated by lower expression of α-smooth muscle actin, collagen I, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1. Anti-ST2 treatment was also associated with reduced hepatic infiltration of CD4⁺ T cells, B cells and M2 macrophages, along with decreased systemic levels of IFN-γ, TNF-α, IL-4, IL-13, IL-17 and IL-1β. ST2 blockade reduced metacestode growth and was associated with attenuated hepatic inflammation and fibrosis, supporting a role for IL-33/ST2 signalling in AE progression. Given the concurrent reduction in parasite burden, further studies are needed to determine whether the anti-fibrotic effects of ST2 inhibition are independent of parasite control.

Chemical composition of calcareous corpuscles in Echinococcus multilocularis protoscoleces.

Alveolar echinococcosis is a life-threatening zoonotic parasitic disease caused by infection of Echinococcus multilocularis larvae. To survive within the host's immune milieu, E. multilocularis has evolved sophisticated immune evasion strategies, including the expression of immunomodulatory proteins that regulate the host immune response. Our previous studies have demonstrated that E. multilocularis calreticulin (EmCRT) possessed strong binding ability to human complement component C1q to inhibit C1q-initiated complement activation and biological functions. To further elucidate the mechanism by which EmCRT mediates C1q inactivation and immune evasion, the precise C1q-binding site on EmCRT was identified and analyzed in this study through expression of overlapping fragments and synthesis of overlapping peptides covering the identified functional fragment. The fragment expression and functional assay narrowed down the C1q-binding site to the EmCRT-S1 fragment located between amino acids 140 and 204 of EmCRT. The precise binding site was further pinpointed to the P5 peptide (EmCRT160-174 aa) by testing the synthetic peptides covering this region. The binding of peptide P5 to C1q markedly suppressed the activation of the C1q-mediated classical complement pathway and C1q-induced neutrophil chemotaxis, production of reactive oxygen species, cathepsin G, and myeloperoxidase. These findings suggest that the C1q-binding P5 peptide of EmCRT may serve as a potential target for the development of vaccines against echinococcosis or therapeutic drugs for complement-associated inflammatory or autoimmune diseases.

Echinococcus multilocularis serine protease inhibitor 1 (EmSPI-1): a highly effective serodiagnostic antigen for alveolar echinococcosis.

Rapid multiplex detection of Echinococcus granulosus and Echinococcus multilocularis using a one-pot RPA-assisted CRISPR-Cas12a/Cas13a assay in a portable multi-tube device.

Echinococcosis, caused by Echinococcus multilocularis (Em) and Echinococcus granulosus (Eg), induces distinct patterns of liver fibrosis. Understanding how protoscoleces (PSCs) from these species interact with hepatic stellate cells (HSCs) is crucial to elucidating the mechanisms of fibrogenesis. In this study, HSC-LX2 cells were co-cultured with Em and Eg PSCs. ELISA was used to quantify collagen-I, α-smooth muscle actin (α-SMA), osteopontin (OPN), and TNF-α levels, while transmission electron microscopy (TEM) examined ultrastructural changes. Both Em- and Eg-derived PSCs significantly increased ECM protein expression, but Em PSCs induced earlier and stronger responses, accompanied by more pronounced structural alterations in HSCs. These findings demonstrate species-specific HSC activation and provide insight into the differential fibrogenic potential of Em and Eg.

Echinococcosis remains a major neglected zoonosis with limited therapeutic options. Thymol and carvacrol have emerged as experimental anti-echinococcal candidates, yet their mechanistic basis, translational relevance, and major limitations including the absence of clinical evidence remain incompletely defined. This review critically evaluates available evidence on their anti-echinococcal activity, examines proposed mechanisms of action, and identifies key barriers that must be addressed for therapeutic development. A substantial body of experimental research has been conducted to evaluate their efficacy, and this narrative review synthesizes and critically examines available evidence regarding their anti-echinococcal activity and proposed mechanisms of action. Available experimental evidence indicates that both isomers exhibit anti-echinococcal activity in in vitro and in vivo models involving Echinococcus granulosus sensu lato and Echinococcus multilocularis. Their effects are mainly associated with tegumental membrane disruption, accompanied by ultrastructural alterations, while evidence supporting apoptosis-like pathways remains preliminary and incompletely validated. Experimental studies indicate synergistic or additive interactions with albendazole, suggesting possible mechanistic complementarity, although the basis of these interactions remains incompletely defined. Although experimental evidence supports membrane-disruptive and anti-echinococcal activity of thymol and carvacrol, current evidence remains largely preclinical and insufficient for therapeutic translation, particularly in the absence of clinical research data. Major uncertainties including poor aqueous solubility, limited bioavailability, lack of intracystic pharmacokinetic data, methodological heterogeneity, and incompletely validated mechanistic pathways remain unresolved. These monoterpenoid phenols should therefore be regarded as promising experimental candidates rather than clinically translatable agents, pending rigorous preclinical validation.

In Serbia, human echinococcosis cases are predominantly diagnosed as the cystic form, caused by Echinococcus granulosus sensu lato (s. l.) complex tapeworms. Alveolar echinococcosis, a more severe disease caused by Echinococcus multilocularis, emerged recently. Domestic dogs have been implicated in the transmission of E. granulosus sensu stricto G1/3 to humans. In an ongoing screening of definitive hosts for the presence of Echinococcus spp. to discover reservoirs of E. multilocularis, fecal samples of hunting (n = 29) and stray dogs (n = 66) were examined. Taeniid eggs were collected using flotation and mesh filtration. The presence of Cyclophyllidea, E. granulosus s. l. and E. multilocularis DNA was detected by specific PCR assays. In total, 60% of the fecal samples contained cyclophyllidean DNA, while Echinococcus spp. DNA was detected in 7.4% of the samples. E. canadensis G6-8,10 DNA was present in 4.2% and E. multilocularis in 3.2% of the samples. E. multilocularis DNA was detected only in stray dog feces, suggesting that strays are a reservoir and possibly an important link in the transmission chain. To the best of our knowledge, this study provides the first molecular identification of Echinococcus spp. in dogs in Serbia.

Introduction: Alveolar echinococcosis (AE) is a rare zoonotic infection caused by Echinococcus multilocularis. In recent years, the number of reported cases has increased across Europe, including Switzerland. Methods: We conducted a retrospective observational study at Lausanne University Hospital (CHUV), including all AE cases diagnosed between 2010 and 2023 within the Departments of Gastroenterology and Hepatology, Visceral Surgery, and Infectious Diseases. Results: Eighty patients with AE were identified. Half of them were male. The mean annual incidence was 0.53 cases per 100,000 population (range, 0.14–0.85), with a statistically significant increase over time in the canton of Vaud (p = 0.037). The median age at diagnosis was 61.5 years, showing a significant downward trend during the study period. At diagnosis, 45 patients (56.3%) were asymptomatic, and 17 (21.3%) were immunosuppressed. Compared with immunocompetent patients, immunosuppressed individuals more often presented asymptomatically, had significantly smaller hepatic lesions, and were more frequently seronegative. Conclusions: This study demonstrates evolving epidemiological patterns of AE in Switzerland, characterized by increasing incidence and a high proportion of asymptomatic cases. Immunosuppressed patients tended to present with milder disease. Our findings highlight the need for strengthened surveillance and support the ongoing efforts to establish a national AE registry.

Inflammatory bowel disease (IBD) manifests as a sustained gastrointestinal condition driven by an ongoing dysregulated immunity, defective mucosal barrier, and microbiota dysbiosis. Parasitic helminths exert immunomodulation chiefly via excretory/secretory proteins (ESP). This study assessed the remedial effects alongside the mechanistic pathways of the ESP derived from Echinococcus multilocularis (Emu-ESP) within a dextran sulfate sodium (DSS)-provoked colitis paradigm in mice. Our results indicated that Emu-ESP treatment significantly mitigated DSS-induced colitis and notably alleviated gut microbiota dysbiosis associated with colitis. Mechanistically, Emu-ESP preserved the gut barrier by enhancing the expression of mucin and epithelial junction genes invivo and reduced organoid injury exvivo with decreased apoptosis and NF-κB signaling. Furthermore, Emu-ESP could promote macrophage polarization toward the M2 phenotype during colitis. In conclusion, the therapeutic effectiveness of Emu-ESP is linked to its regulation of pathways that suppress inflammation, maintain intestinal barrier function, and prevent microbiota dysbiosis, suggesting it as a promising treatment for intestinal inflammatory disorders.

BackgroundThe C-C motif chemokine ligand 2 (CCL2)–C-C chemokine receptor type 2 (CCR2) signaling axis is critically involved in angiogenesis and cellular invasion; however, the therapeutic potential of its targeted blockade on alveolar echinococcosis (AE) remains largely unexplored. This study aimed to determine whether RS504393, a selective CCR2 antagonist, can inhibit the progression of AE by blocking the CCL2–CCR2 axis and modulating its downstream pathogenic mechanisms.MethodsIn this study, bioinformatics analysis of the GSE124362 dataset was combined with molecular docking simulations. Liver tissues from patients with AE and from Echinococcus multilocularis-infected mice were examined through histopathological and immunohistochemical staining methods. Pathological alterations and parasitic load were assessed by Western blot and quantitative real-time PCR (RT-PCR). An in vitro co-culture model involving Echinococcus multilocularis protoscolex-stimulated endothelial progenitor cells (EPCs), JS1 hepatic stellate cells (JS1) and RAW264.7 macrophages was established to evaluate angiogenesis, fibrogenic activity and macrophage polarization.ResultsIntegrated bioinformatics and molecular docking analyses revealed CCL2–CCR2 overexpression and high-affinity binding during AE progression. Treatment with RS504393 significantly attenuated hepatic fibrosis, suppressed phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) pathway activation and reduced M2 macrophage polarization in central lesion tissue of E. multilocularis-infected mice . In vitro, RS504393 inhibited angiogenesis driven by EPCs, induced apoptosis in JS1 cells, and redirected macrophage polarization from the M2 towards a more anti-parasitic phenotype.ConclusionsCCR2 blockade with RS504393 attenuates AE progression by inhibiting PI3K-AKT signaling, suppressing angiogenesis and inducing stellate cell apoptosis, collectively reducing hepatic fibrosis and parasitic burden. Our findings provide a mechanistic rationale for repurposing CCR2 antagonists as a novel host-directed therapeutic strategy for echinococcosis.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-026-07306-3.

This study demonstrates that compound Biejia-Ruangan tablet (CBRT) alleviates Echinococcus multilocularis (E.m.)-induced hepatic fibrosis by reactivating the suppressed STING-TBK1 signaling pathway in the chronic infection stage, thereby reprogramming macrophage polarization from a pro-fibrotic M2 phenotype toward an M1 phenotype. This work identifies CBRT as a promising immunomodulatory agent that can reverse the Th2/M2-dominated immunosuppressive microenvironment, providing a novel therapeutic strategy for AE and potentially other infection-associated fibrotic diseases. However, further research is required to validate the translational efficacy of CBRT and to define the precise therapeutic window for STING-TBK1 modulation in human fibrotic disorders.

Alveolar echinococcosis (AE), caused by Echinococcus multilocularis (E. multilocularis), exhibits tumor-like, invasive growth in the liver. Unlike cystic echinococcosis, AE lesions are often bordered by loose fibrosis rather than a dense fibrotic capsule. The mechanism by which the parasite attenuates this host fibrotic response remains unknown. We performed small RNA sequencing of parasite-derived exosomes (EmV-EXOs) and multi-step screening to identify functional miRNAs. The candidate was validated via bioinformatics, dual-luciferase assays, and in vitro studies in hepatic stellate cells (HSCs). An adeno-associated virus (AAV6)-delivered Tough Decoy (TuD) RNA was used for in vivo inhibition in a murine model. emu-miR-745-3p was identified as an exosomal miRNA enriched in EmV-EXOs. It is delivered to HSCs and directly targets the 3' UTR of dipeptidyl peptidase-4 (DPP4), a pro-fibrotic regulator. Downregulation of DPP4 suppressed HSCs activation, reducing expression of α-SMA, COL1A1, and TIMP1. In vivo inhibition of emu-miR-745-3p enhanced perilesional fibrosis, promoted a thicker fibrous capsule, and significantly reduced parasitic lesion burden. E. multilocularis employs exosomal emu-miR-745-3p to attenuate host fibrotic encapsulation, facilitating invasive growth. The emu-miR-745-3p/DPP4 axis is a critical determinant of AE pathology and a potential target for novel anti-fibrotic therapeutics.

EmCRT is a calreticulin secreted by Echinococcus multilocularis during its infection in host, playing an important role in evading host immune attack as a survival strategy. Our previous study has demonstrated that recombinant EmCRT (rEmCRT) was able to bind to C1q and lectin to interfere with host classical and lectin complement activation pathway, respectively. However, the C1q-binding site on EmCRT and the associated immune evasion mechanism remain unknown. In this study, the C1q-binding site on EmCRT was determined through molecular docking analysis and fragment expression to be localized to the S-domain (EmCRT-S) between Lys140 at the N-domain and Gln292 at the P-domain. The recombinant EmCRT-S protein (rEmCRT-S) was subsequently expressed in bacteria. Functional analysis confirmed that rEmCRT-S was able to bind to human C1q and inhibit C1q-initiated complement activation at the similar level to the full-length rEmCRT, resulting in the reduction in C4b/C3b deposition and antibody-sensitized sheep red blood cell hemolysis. Furthermore, rEmCRT-S binding to C1q suppressed THP-1-derived macrophage chemotaxis and ROS generation. Given that the identified functional domain EmCRT-S provides similar complement regulatory functions to the full-length EmCRT, this domain is a more feasible and practical target for vaccine development against E. multilocularis infection or for inflammatory and autoimmune diseases.

Alveolar echinococcosis (AE), caused by Echinococcus multilocularis larvae, is a severe zoonotic disease mimicking tumors, primarily affecting the liver with high mortality if untreated. Host immunity plays a pivotal role, shifting from Th1/Th17-mediated clearance to Th2/Treg-driven tolerance, enabling parasite survival. Liver macrophages, including Kupffer cells, polarize towards M2 phenotype under parasite antigens (e.g., phytic acid, exosomes), promoting immunosuppression, fibrosis, and T cell exhaustion via IL-10/TGF-β. This reshapes the tumor-like immune microenvironment with M2 macrophages recruiting Tregs, suppressing NK/DC functions, and fostering angiogenesis/fibrosis. Current treatment remains centered on surgery and benzimidazole therapy, both of which have notable limitations. Experimental immunomodulatory strategies, drug repurposing approaches, and targeted delivery systems may offer future therapeutic opportunities, but these concepts remain largely preclinical, unproven in AE, and require careful evaluation for safety and efficacy.

The movement of dogs from continental Europe to the UK poses a growing public health threat due to the associated risk of disease incursions. Current legislation is insufficient to address the risks and pre-import control measures are focused only on rabies virus and the fox tapeworm Echinococcus multilocularis. We conducted a scoping review to summarise the major zoonotic pathogens and vectors associated with imported dogs (including rabies virus, Brucella canis and exotic tick species), and explore their potential to become established in the UK. Gaps in existing research and surveillance are highlighted, and potential measures to strengthen control are discussed. Only English-language sources were included in the literature search. Data on imported dogs and their disease burden are limited. Raising awareness of the risks among veterinary staff-who play a central role in recognising, managing and preventing imported zoonoses-is integral to a One Health approach.

Both alveolar echinococcosis and cystic echinococcosis are zoonotic diseases affecting thousands of humans in Europe annually. Five Echinococcus species are known to be endemic in Europe, but with varying relative importance in humans and animals. In order to identify these Echinococcus species from tissue samples, an RFLP-PCR method was developed and validated in accordance with the French quality standards for molecular diagnostics (NF U47-600-2). Amplification by PCR of about 846 bp from the 12S ribosomal RNA gene was followed by a single digestion step with three restriction enzymes (AccI, DdeI and HinfI). The RFLP-PCR method distinguishes Echinococcus multilocularis, E. granulosus sensu stricto, E. equinus, E. ortleppi and E. canadensis in six specific profiles composed of two or three bands. Each profile is specific for the species, except an additional specific profile from genotype G8 of E. canadensis. Species from other genera of the Taeniidae family correspond to two other types of profiles. Additionally, in silico analyses predicted three specific additional profiles for the other four Echinococcus species. The limit of detection of the method including DNA extraction is estimated to be a minimum of 1,000 copies. Total specificity and sensitivity were obtained by testing a panel of 73 and 79 DNA samples previously identified by sequencing. This RFLP-PCR technique is cost-effective, simple and rapid to implement, making it suitable for use in large epidemiological studies, as well as for single diagnostic purposes targeting tissue samples, such as cysts or worms, without requiring sequencing.

Echinococcus multilocularis is a zoonotic cestode that uses canids as definitive hosts and rodents as intermediate hosts. In humans, this parasite is the causative agent of alveolar echinococcosis. Recently, its range has been expanding across the Northern Hemisphere, and it is increasingly detected in wild canids, domestic dogs, and humans across Canada and the United States. While this expansion has been documented in isolated studies across the continent, a lack of routine sampling in wildlife hinders our ability to anticipate and mitigate further spread of E. multilocularis. We confirmed the presence of E. multilocularis in Washington State, USA, using a combination of morphological and molecular techniques across carcasses and field-collected scats of coyotes (Canis latrans), this region’s most common wild canid. Morphological identification of adult worms was confirmed by next-generation sequencing. Over a third of all samples tested positive for E. multilocularis when all methodologies were combined. Sequencing revealed a haplotype of E. multilocularis matching a documented haplotype originally of European origin in British Columbia, Canada. Our study provides the first confirmation of E. multilocularis in a wild host on the west coast of the contiguous United States and provides additional haplotype information crucial to tracking the geographical expansion of the parasite. We also provide a new next-generation sequencing primer targeting cestodes of canids. The difference in amplification between intestinal and fecal samples suggests that non-invasive fecal sampling using DNA metabarcoding - a popular method of helminth surveillance - may lead to underestimation of prevalence, hindering control measures. The global significance of these findings extends beyond North America; E. multilocularis is a major public health concern in Europe and Asia, where alveolar echinococcosis is increasingly diagnosed in humans. Our study highlights the urgent need for increased surveillance and improved diagnostic strategies worldwide, particularly in regions with significant human-wildlife contact.

Alveolar echinococcosis (AE) is a lethal zoonosis caused by infiltrative growth of the metacestode larva of the tapeworm Echinococcus multilocularis in host organs. We previously showed that the Echinococcus metacestode is an evolutionarily unique, broadly posteriorized tissue, leading us to hypothesize that canonical WNT (cWNT) signalling, which patterns the body axis across metazoans, might be critical for metacestode formation. Here, we report effective RNAi-mediated knockdown of the E. multilocularis β-catenin gene (bcat-1), the central effector of cWNT signalling, in a primary parasite cell culture system that produces metacestode vesicles. bcat-1(RNAi) cultures were markedly impaired in vesicle formation, exhibited stem-cell hyperproliferation, and displayed changes in muscle-fibre organisation. Genome-wide transcriptomics revealed a general anteriorization of gene expression, and in situ hybridization showed an overproduction of cells expressing head-inducing factors such as sfrp upon bcat-1 knockdown. Conversely, metacestode-specific genes including the tegumental factors muc-1, TNFR, and antigen B as well as the posterior marker post2b were significantly downregulated, consistent with the observed vesicle-formation defects. In situ analyses further identified anterior markers frizzled-10, nou-darake, notum, and follistatin that were overexpressed in bcat-1(RNAi) cultures and localized to the future anterior pole at the earliest stages of protoscolex formation. Together, these findings establish a central role for cWNT signalling in directing Echinococcus body-axis formation and the posteriorization events driving metacestode growth within the host, providing insight into asexual parasite proliferation mediated by this biologically unique larval stage and pointing to potential targets for chemotherapy against AE.