Alveolar echinococcosis (AE) is a serious zoonotic parasitic disease caused by the larvae of Echinococcus multilocularis. Given the limitations of current medications, there is an urgent necessity for the development of novel pharmacological treatments for AE. Azithromycin, a macrolide antibiotic, exhibits both antibacterial and antiparasitic effects. This study aimed to investigate its effects on the viability of E. multilocularis protoscoleces (EmPSCs) and to elucidate the underlying mechanisms. Ultrastructural changes were examined using scanning and transmission electron microscopy. Glucose assays, reactive oxygen species (ROS) assays, mitochondrial membrane potential (MMP) assays, and Western blot were used to evaluate the in vitro effects of azithromycin on EmPSCs. Our results demonstrated that azithromycin treatment significantly decreased the viability of EmPSCs and caused pronounced ultrastructural damage. Mechanistically, it significantly downregulated glucose transporter 1 (GLUT1) and protein kinase B (AKT) signaling, elevated ROS levels (P < 0.001), and decreased MMP. Furthermore, Azithromycin treatment significantly elevated the expression of the DNA damage marker γ-H2AX (P < 0.001), while also increasing lipid peroxidation and Fe[2+] levels, depleting GSH content, and downregulating GPX4 and DHODH expression(P < 0.001). Collectively, azithromycin may exert multiple effects on EmPSCs in vitro, encompassing glucose metabolic disruption, oxidative stress, and ferroptosis.

Here, we review equine alveolar echinococcosis (AE), focusing on its epidemiology, clinical and pathological features, and diagnostic challenges, through comparisons with AE in other host species. AE is a zoonotic disease caused by the larval form of the Echinococcus multilocularis tapeworm. Horses are considered dead-end hosts, in which AE infection can be detected at slaughter, highlighting their potential as sentinel animals in endemic areas. Equine AE is typically asymptomatic and is generally only found incidentally during meat inspection or necropsy. Standard diagnosis involves histopathology and conventional polymerase chain reaction (PCR) testing. Epidemiological surveys in Japan, Poland, and Canada have reported larval E. multilocularis detection rates ranging from 1.5-18.8%, where infections manifest as macroscopic grayish-white solid liver nodules in slaughtered horses. Molecular phylogenetic analysis targeting the mitochondrial cytochrome b(cob) gene has revealed that parasites isolated from horses in Japan and Canada belong to distinct haplogroups, indicating utility for elucidating the geographical genetic characteristics of larval E. multilocularis infection in horses. Isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) have demonstrated equivalent or superior sensitivity and specificity for detecting equine AE and may represent novel rapid genetic diagnostic tools as alternatives to PCR testing for rapid gene-based detection. These techniques will contribute to early detection of AE, especially in locations where specialized laboratory equipment is limited (e.g., abattoirs). Continued research on equine AE will thus greatly contribute to elucidating the environmental distribution of E. multilocularis and supporting the control of zoonotic disease.

Alveolar echinococcosis (AE) is a severe zoonotic parasitic disease caused by the larval stage of Echinococcus multilocularis, for which early and accurate diagnosis remains challenging. In this study, we targeted E. multilocularis thioredoxin peroxidase (EmTPx), a highly abundant excretory/secretory protein conserved across the genus Echinococcus, for diagnostic development. Monoclonal antibodies (mAbs) against EmTPx were generated, and two linear B‑cell epitopes, 174HPGSK178 and 189SFMSS193, were identified. Both epitopes are strictly conserved within Echinococcus but markedly divergent from mammalian host sequences. Structural modeling revealed that the epitopes are surface‑exposed, with 174HPGSK178 located in a flexible loop and 189SFMSS193 adjacent to an α‑helix, conformations favorable for antibody recognition. Two ELISA methods were established using the epitope‑specific mAbs. The blocking ELISA showed moderate diagnostic performance. More importantly, the sandwich ELISA enabled direct detection of EmTPx antigen in fecal samples of intermediate hosts, revealing a detection rate of 85% in mouse feces at an early infection stage. With a linear range of 0-250 ng/mL, a cutoff of 0.1724, and intra‑ and inter‑assay precision below 10%, this sandwich ELISA demonstrated robust analytical performance. In conclusion, this proof-of-concept study identifies two evolutionarily conserved and structurally exposed EmTPx epitopes and demonstrates their potential utility for the development of monoclonal antibody-based approaches for non-invasive detection of echinococcosis.

Echinococcus multilocularis and Echinococcus granulosus sensu lato are the causative agents of two major zoonotic diseases, alveolar echinococcosis and cystic echinococcosis, respectively, and are ranked among the four most important foodborne parasites worldwide. Although interest in food contamination by taeniid eggs is increasing, the estimation of the number of eggs present in food items is still lacking. Using E. multilocularis eggs isolated from fecal samples of experimentally infected red foxes, an average of 5261 (±1253) mitochondrial DNA copies per egg was estimated by digital PCR (dPCR). Based on this estimation, 47 taeniid DNA samples previously obtained from different food items and identified as positive for E. multilocularis, E. granulosus sensu stricto or Taenia species, were submitted to dPCR to estimate the number of eggs present. In 95.7% of the samples, the contamination was estimated to be caused by one to five eggs, with only one egg in 83.0% of the samples. As detection of such low-level contamination requires sensitive detection methods, a performance comparison between microscopy and molecular methods was conducted using 15 pellets obtained from lettuce wash residues spiked with 0 to 63 E. multilocularis eggs. Two operators performed blinded microscopy, followed by DNA extraction from the examined pellets for real-time PCR detection and then an estimation of the number of eggs by dPCR. A higher sensitivity was obtained with real-time PCR (88.5%) compared to microscopy (40%) in samples containing one to five eggs. The correlation between the number of eggs spiked into the samples and the estimated egg number was strong using dPCR (r = 0.99, p < 0.01), while only moderate with microscopy (r = 0.67, p < 0.05). Estimation of the number of taeniid eggs in food samples by dPCR provides the first quantitative basis for exposure assessment and is important for evaluating the risk of human foodborne infections, with future work needed to address egg viability.

Alveolar echinococcosis (AE), caused by the metacestode stage of Echinococcus multilocularis, is a highly lethal zoonosis. Its insidious progression frequently precludes diagnosis until advanced stages, and the paucity of effective early detection tools remains a critical impediment to disease control. In this study, we aimed to evaluate the diagnostic utility of EmCIP2A as a target antigen and develop monoclonal antibody-based tools for the serological and non-invasive detection of E. multilocularis infection. Six monoclonal antibodies (mAbs) were generated against EmCIP2A, a germinative cell-specific marker of the parasite. Isotyping identified 1F3C8 and 2F7G3 as IgG1, and 1G9E4, 3F1G4, 4B1C6, and 5A7C11 as IgG2b, all bearing kappa light chains. Western blotting and indirect immunofluorescence confirmed that all mAbs specifically recognize both recombinant and native EmCIP2A. Four linear B-cell epitopes were delineated with 1F3C8/5A7C11 targeted [229]MADLSLTEER[238], 4B1C6 bound [384]HHSEATLNRL[393], 2F7G3 recognized [432]AEVEAKREAL[441], and 1G9E4/3F1G4 reacted with [574]TGNRVQPANA[583]. Based on epitope and affinity screening, a blocking ELISA employing HRP-1F3C8 was established (AUC = 0.8494; sensitivity, 90.63%; specificity, 72.73%). In addition, a double-antibody sandwich ELISA using 1F3C8 as capture antibody and HRP-2F7G3 as detection antibody was further developed, displaying excellent linearity (1-1000 ng/mL, R[2] = 0.9973); preliminary fecal testing yielded detection rates of 70% in infected mice and 50% in infected dogs, confirming its utility for active infection surveillance across intermediate and definitive hosts. Collectively, the two ELISA systems provide complementary modalities, serological screening and non-invasive fecal antigen detection, which together expand the diagnostic repertoire against E. multilocularis infection.

Alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis, is a severe zoonosis requiring prolonged anthelmintic therapy. Albendazole (ABZ) remains the treatment of choice; however, its poor aqueous solubility and low systemic bioavailability limit therapeutic efficacy and necessitate prolonged high-dose regimens. Improving the delivery of existing antiparasitic drugs therefore represents a valuable therapeutic strategy. This study evaluated the in vivo chemoprophylactic and therapeutic efficacy of an innovative nanomedicine formulation comprising ABZ encapsulated in poly(hydroxybutyrate) (PHB) nanoparticles synthesized by electrospraying. The resulting formulation demonstrated high reproducibility, adequate drug loading, and stable physicochemical characteristics suitable for in vivo evaluation. Efficacy was assessed in an experimental murine model of intraperitoneal alveolar echinococcosis using chemoprophylactic and therapeutic protocols. In the chemoprophylactic study, ABZ-loaded PHB nanoparticles significantly reduced cyst weight compared with the untreated control group and induced extensive ultrastructural damage to the germinal layer, outperforming the ABZ suspension. In the therapeutic protocol, the polymeric nanocarrier system significantly impaired metacestode tissue viability relative to the untreated control. Notably, despite the lack of a statistically significant difference in the cyst weights compared to the ABZ suspension, the nanoparticle treatment induced far more pronounced ultrastructural alterations in the germinal layer. This severe damage, characterized by a marked reduction in germinative cells, effectively compromised the regenerative capacity and proliferative potential of the metacestode tissue. In conclusion, the electrosprayed PHB nanocarrier system represents a promising biopharmaceutical strategy to optimize ABZ delivery, enhancing its therapeutic performance against experimental alveolar echinococcosis without requiring increased drug dosages.

Mesenchymal stem cells (MSCs) exhibit potent immunomodulatory properties, yet their precise mechanisms in alleviating Echinococcus multilocularis-induced hepatic inflammation and fibrosis in Alveolar Echinococcosis (AE) remain elusive. This study aimed to elucidate the immunoregulatory mechanisms underlying MSCs therapy in AE. Using an AE mouse model, we demonstrated that MSCs administration significantly ameliorated liver histopathology, attenuated fibrosis, and restored liver function. Molecular analyses revealed that AE infection promoted a pro-inflammatory profile (elevated RORγt and IFN-γ) while suppressing regulatory factors (FOXP3, TGF-β1, IL-10, CNTN1, and Gilz), effects that were robustly reversed by MSCs treatment. In vitro, co-culture of CNTN1-knockdown MSCs with E. multilocularis antigen-stimulated CD4[+] T cells demonstrated that CNTN1 deficiency impaired MSCs-mediated immunoregulation, decreased IL-10 secretion, and disrupted the Th17/Treg balance. Mechanistically, dual-luciferase reporter assays confirmed that CNTN1 directly activates the Gilz promoter. Collectively, our findings identify the CNTN1/Gilz signaling axis as an essential mediator of MSCs immunomodulation, providing novel mechanistic insights into the therapeutic efficacy of MSCs against E. multilocularis-induced hepatic injury.

Parasitic flatworms, including cestodes and trematodes, are covered by a specialized syncytial tegument that mediates nutrient uptake and host-parasite interactions. While the tegument of trematodes has been extensively characterized, its molecular composition in cestodes remains largely unknown. In this work, we performed a comparative proteomic analysis of the tegument of three cestode species, including larval and adult stages: Hymenolepis microstoma, Mesocestoides corti (syn. M. vogae) and the human pathogen Echinococcus multilocularis. Using stringent enrichment criteria relative to whole-worm extracts, we identified hundreds of tegument-enriched proteins in each species. Comparative analyses revealed a conserved core of tegumental proteins shared among all three species, including members of the Tegument Allergen-Like (TAL) family, vesicular trafficking components and calcium-sensing proteins, and identified candidates for nutrient uptake activities such as glucose and nucleoside transporters. Further comparative analyses revealed a set of shared tegumental proteins with the trematode Schistosoma mansoni, including conserved proteins that are specific to parasitic flatworms, supporting the existence of a conserved ancestral tegumental proteome. Finally, we confirmed tegumental expression of several genes in H. microstoma and E. multilocularis, and demonstrated regionally restricted gene expression among tegumental cytons, suggesting functional specialization within the syncytial tegument. Altogether, these results reveal an evolutionarily conserved composition of the tegument of parasitic flatworms, providing a foundation for future work targeting this critical host-parasite interface.