Echinococcus Multilocularis Infection Research Articles

SCD155 as a potential marker for diagnosing the vascular invasion in hepatic alveolar echinococcosis

BackgroundAlveolar Echinococcosis (AE) is a malignant zoonotic disease caused by Echinococcus multilocularis infection. Considering whether the lesion is accompanied by vascular invasion (VI) is crucial for treatment strategies. A cost-effective and convenient clinical diagnostic method is urgently needed to supplement current techniques. Consequently, we detected soluble CD155 (sCD155) as a potential biomarker for diagnosing VI in hepatic alveolar echinococcosis (HAE). MethodsBlood samples were from 42 AE patients and 49 healthy controls (HCs). Based on the computed tomography (CT) and contrast-enhanced CT, AE patients were further categorized into HAE with VI (VIAE; 27 cases) and HAE without VI (NVAE; 15 cases). The sCD155 concentration was measured by an enzyme-linked immunosorbent assay (ELISA). Correlations between sCD155 expression levels and clinicopathological features of AE patients were analyzed using SPSS and GraphPad Prism software. ResultsThe sCD155 concentrations in AE patients were significantly higher than in HCs. The serum sCD155 level significantly differed between the VIAE and NVAE groups. The univariate analysis showed that VI of AE was significantly correlated with the sCD155 level when the sCD155 was greater than 11 ng/mL. After adjusting for potential confounding factors, the multivariable analysis showed that sCD155 had an independent effect on VI of HAE. The receiver operating characteristic (ROC) curve showed that sCD155 could differentially diagnose VI of HAE at the cut-off value of 11.08 ng/mL with an area under the curve (AUC) value of 0.75. The sensitivity and specificity were 74.07 % and 66.67 %, respectively; the positive and negative predictive values were 74.07 % and 60.00 %, respectively. ConclusionThe sCD155 could be a VI biomarker for HAE. Elevated sCD155 levels are indicative of an increased likelihood of concomitant VI in HAE patients, necessitating a thorough evaluation of vascular impairment and the formulation of individualized management strategies.

Study on the mechanism of miRNAs on liver injury in the condition of Protoscocephalus alveolarus transhepatic portal vein infection.

To explore the role of miRNA in liver damage caused by Echinococcus multilocularis infection. Six female C57BL mice were randomly divided into two groups, the control group and the infection group. Mice in the control group were injected with 100 μL PBS through the hepatic portal vein, and mice in the infection group were infected with E. multilocularis via the hepatic portal vein to establish a mouse model of infection. Small RNA sequencing was performed for detecting the expression of miRNAs in the liver of mice infected with 2000 E. multilocularis after 3 months of infection, screen out miRNAs related to liver damage, and verify by RT-PCR. Seventy-one differentially expressed miRNAs were found in the liver in comparison with control, and a total of 36 mouse miRNAs with |FC| >0.585 were screened out, respectively. In addition, Targetscan (V5.0) and miRanda (v3.3a) software were used to predict differential miRNAs target genes and functional enrichment of target genes. Functional annotation showed that "cytokine-cytokine interaction," "positive regulation of cytokine production," "inflammatory response," and "leukocyte activation" were enriched in the liver of E. multilocularis-infected mice. Moreover, the pathways "human cytomegalovirus infection," "cysteine and methionine metabolism," "Notch signaling pathway," and "ferroptosis" were involved in liver disease. Furthermore, four miRNAs (mmu-miR-30e-3p, mmu-miR-203-3p, mmu-miR-125b-5p, and mmu-miR-30c-2-3p) related to liver injury were screened and verified. This study revealed that the expression profiling of miRNAs in the livers was changed after E. multilocularis infection, and improved our understanding of the transcriptomic landscape of hepatic echinococcosis in mice.

Dynamic observation on capillarization of liver sinusoidal endothelial cells induced by Echinococcus multilocularis infection

To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-, 2- and 4-week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate-buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post-infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin-eosin (HE) staining, and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson's trichrome staining-positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf was quantified using real-time fluorescence quantitative PCR (qPCR) assay. Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post-infection. Semi-quantitative analysis of Masson's trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection, and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection, which spread to surrounding tissues. Semi-quantitative analysis revealed significant differences in α-SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α-SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4-week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1-[(1.22 ± 0.48)/μm2 and [(3.05 ± 0.91)%] and 2-week infection groups [(3.47 ± 0.10)/μm2 and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1-[(180.80 ± 16.42) nm] and 2-week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin-1 (F = 153.100, P < 0.001), Stabilin-2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.

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

BackgroundAlveolar 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.MethodsIn 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.ResultsThis 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 β 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 β-overexpressed KCs.ConclusionsThese results demonstrated that miR-374b-5p probably regulated the expression of inflammatory factors via C/EBP β/NF-κB signaling. This finding is helpful to explore the mechanism of inflammation regulation during E. multilocularis infection.Graphical

The expression of CTLA-4 in hepatic alveolar echinococcosis patients and blocking CTLA-4 to reverse T cell exhaustion in Echinococcus multilocularis-infected mice.

Alveolar echinococcosis (AE) is a zoonotic parasitic disease caused by the infection of Echinococcus multilocularis (E. multilocularis) larvae. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) produces inhibitory signals and induces T cell exhaustion, thereby inhibiting the parasiticidal efficacy of the liver immune system. Therefore, the purpose of this study is to explore how T-cell exhaustion contributes to AE and whether blocking CTLA-4 could reverse T cell exhaustion. Here we discovered that the expression of CTLA-4 was increased in the infiltrating margin around the lesion of the liver from AE patients by using western blot and immunohistochemistry assay. Multiple fluorescence immunohistochemistry identified that CTLA-4 and CD4/CD8 molecules were co-localized. For in vitro experiments, it was found that the sustained stimulation of E. multilocularis antigen could induce T cell exhaustion, blocking CTLA-4-reversed T cell exhaustion. For in vivo experiments, the expression of CTLA-4 was increased in the liver of E. multilocularis-infected mice, and the CTLA-4 and CD4/CD8 molecules were co-localized. Flow cytometry analysis demonstrated that the percentages of both CD4+ T cells and CD8+ T cells in the liver and peripheral blood were significantly increased and induced T exhaustion. When the mice were treated with anti-CTLA-4 antibodies, the number and weight of the lesions decreased significantly. Meanwhile, the flow cytometry results suggested that blocking CTLA-4 could effectively reverse T cell exhaustion and reactivate immune function. Our work reveals that blocking CTLA-4 could effectively reverse the T cell exhaustion caused by E. multilocularis and could be used as a novel target for the treatment of AE.

Molecular phylogenetic analysis of Echinococcus multilocularis from horses raised in Canada or Japan, using mitochondrial cytochrome b gene–targeted PCR

Alveolar echinococcosis is a zoonotic disease caused by a larval-stage Echinococcus multilocularis infection. Geographical haplotyping targeting the parasite's mitochondrial cytochrome b (cob) gene has been reported for isolates from definitive and intermediate hosts (wild canids and rodents); however, there are limited reports on strain typing for the dead-end host, the horse, which could act as a sentinel for E. multilocularis. Accordingly, we investigated the diversity of E. multilocularis in isolates obtained from slaughtered Japanese and Canadian horses originating from the Iburi and Hidaka regions in Hokkaido and from Alberta, respectively, with PCR and haplogroup analyses targeting cob gene sequences obtained. Seventy horses were diagnosed with alveolar echinococcosis based on histopathology and cob-gene PCR testing. The E. multilocularis detected in these horses was classified as either an Asian (for Hokkaido-raised horses) or a European (for Alberta-raised horses) haplogroup, based on the obtained cob-gene sequence analysis. In addition, haplotype network analysis revealed that E. multilocularis isolated from Hokkaido-raised horses is highly homologous to Kazakhstan isolates, and E. multilocularis isolated from Alberta-raised horses is highly homologous to Austrian isolates. The results of this study suggest that cob-gene-targeted PCR analysis could be useful for the geographical genetic characterization of E. multilocularis isolated from horses.

Omics-based investigation of pathological liver injury induced by Echinococcus multilocularis infection in mice

BackgroundAlveolar echinococcosis (AE) can cause severe liver injury and be fatal if left untreated. Currently, there are no effective therapeutic options for AE-induced liver injury. Therefore, by exploring the changes of gene proteins in mice with damaged liver, we attempted to identify the key molecules of liver damage, and provide data that will enable the development of drugs targeting hepatic AE. MethodsBALB/c mice were inoculated with protoscoleces via the hepatic portal vein. Three months later, B-ultrasound examination and Hematoxylin—eosin (H&E) staining were used to confirm liver damage in mice. RNA sequencing and Liquid chromatography–mass spectrometry (LC-MS) were used to screen differentially expressed molecules associated with liver damage through bioinformatics, and Quantitative Real-Time PCR (qRT-PCR) was used to verify their expression. ResultsB-ultrasound examination showed liver lesions in the infected group, and H&E staining showed liver inflammation, fibrosis and liver necrosis. RNA sequencing and LC-MS results showed changes in the levels of more than 1000 genes and proteins, with upregulation of immune and inflammation pathways. By contrast, the downregulated genes and proteins were mostly involved in various metabolic reactions. Correlation analysis was conducted between the transcriptome data and proteome data. The results revealed 240 differentially expressed genes, of which 192 were upregulated, and 48 were downregulated. Many of these genes were involved in metabolic reactions, such as Catalase (Cat), fatty acid synthase (Fasn), and IL-16 genes, which may have relevance to liver injury. The results of qRT-PCR were consistent with those of bioinformatics analysis. ConclusionsThe mechanisms of liver injury in mice infected with Echinococcus multilocularis are complex, involving abnormal metabolism, oxidative stress, inflammatory response, and many other factors. This study provides the data for preliminary exploration for the development of targeted therapies against AE.

Diagnosis of Hepatic Alveolar Echinococcosis with Massive Peritoneal Seeding Treated Surgically- A Case Report

The diagnosis of Alveolar Echinococcosis is often a difficult diagnosis due to its low incidence and a long asymptomatic incubation period. We report a case of a 32-year-old man with an uncommon location of Alveolar Echinococcosis. He presented abdominal pain, conjunctival icterus and dark urine. Abdominal ultrasonography, CT scan and MRI demonstrated the presence of a hepatic lesion and a pararectal mass associated with peritoneal and omental implants. The lesions showed perilesional hypermetabolism on 18F-fluorodeoxiglucose (FDG) positron emission tomography magnetic resonance imaging (PET/MRI). Para-rectal biopsy, serological test, and sample taken during exploratory laparoscopy all suggested Echinococcus multilocularis infection that was later confirmed by PCR test. The patient was treated with Albendazole and underwent curative-intent twostep surgery by right-extended hepatectomy, resection of all peritoneal implants and total mesorectal excision. Thirty months after initial surgery, the patient is doing well and is followed up with MRI and 18F-FDG-PET without morphological signs of recurrence. This case illustrates the capacity of alveolar echinococcosis to affect different organs by infiltration or metastatic dissemination and highlights the role of 18F-FDG-PET/MRI in diagnosis and disease monitoring.

Immunoprotective effect and mechanism of rEg.P29 against CD4 + T cell-deficient mice with Echinococcus multilocularis infection.

Alveolar echinococcosis (AE) is a zoonotic parasitic disease caused by infection with the larval stage of Echinococcus multilocularis and a major challenge to human public health. Vaccines are the most effective way to prevent and control infectious diseases. We previously revealed that the Echinocuccus granulosus recombinant protein P29 is a good vaccine candidate against E. granulosus. However, the protective and immunological mechanism of rEg.P29 against E. multilocularis remain unclear. In this study, CD4 + T cell-deficient mice are transferred with spleen CD4 + T cells isolated from wild-type mice and subjected to rEg.P29 immunization, and then these immunized mice are infected with E. multilocularis. The cyst inhibition rate is calculated by weighing the body and cyst weights. The level of antibody is detected by ELISA. Flow cytometry is used to detect the level of IFN-γ production by CD4 + T and CD8 + T cells. The cytokines in culture supernatant are detected by ELISA. The expressions of CD44 and CD62L on memory T cells are determined by flow cytometry. The results show the cyst inhibition rate is 41.52% after adoptive transfer of CD4 + T cells. Furthermore, the levels of IgG, IgM, IgA and IgE in serum are significantly increased compared with those in the PBS group. The IFN-γ-secretion by CD8 + T cells and the level of IFN-γ in culture supernatant are obviously increased; and the number of CD4 + T cells is increased, but the number of IFN-γ producing CD4 + T cells has no significant difference compared with PBS group. In addition, the number of CD44 +CD62L ‒CD8 + memory T cells in the spleen is significantly increased, while the number of CD44 ‒CD62L + CD8 + memory T cells is not significantly altered. Collectively, rEg.P29 can alleviate E. multilocularis infection by inducing humoral immune responses and CD8 + T cell responses.

Global and local drivers of Echinococcus multilocularis infection in the western Balkan region

The cestode, Echinococcus multilocularis, is one of the most threatening parasitic challenges in the European Union. Despite the warming climate, the parasite intensively spread in Europe's colder and warmer regions. Little is known about the expansion of E. multilocularis in the Balkan region. Ordinary least squares, geographically weighted and multi-scale geographically weighted regressions were used to detect global and local drivers that influenced the prevalence in red foxes and golden jackals in the southwestern part of Hungary. Based on the study of 391 animals, the overall prevalence exceeded 18% (in fox 15.2%, in jackal 21.1%). The regression models revealed that the wetland had a global effect (β = 0.391, p = 0.006). In contrast, on the local scale, the mean annual precipitation (β = 0.285, p = 0.008) and the precipitation seasonality (β = − 0.211, p = 0.014) had statistically significant effects on the infection level. The geospatial models suggested that microclimatic effects might compensate for the disadvantages of a warmer Mediterranean climate. This study calls attention to fine-scale analysis and locally acting environmental factors, which can delay the expected epidemic fade-out. The findings of our study are suggested to consider in surveillance strategies.

Age-dependent relationships among diet, body condition, and Echinococcus multilocularis infection in urban coyotes.

Urban coyotes (Canis latrans) in North America increasingly exhibit a high prevalence of Echinococcus multilocularis, a cestode of recent and rising public health concern that uses rodents as intermediate hosts and canids as definitive hosts. However, little is known about the factors that drive the high urban prevalence of this parasite. We hypothesized that the diet of urban coyotes may contribute to their higher E. multilocularis infection prevalence via either (a) greater exposure to the parasite from increased rodent consumption or (b) increased susceptibility to infection due to the negative health effects of consuming anthropogenic food. We tested these hypotheses by comparing the presence and intensity of E. multilocularis infection to physiological data (age, sex, body condition, and spleen mass), short-term diet (stomach contents), and long-term diet (δ13C and δ15N stable isotopes) in 112 coyote carcasses collected for reasons other than this study from Edmonton, Alberta and the surrounding area. Overall, the best predictor of infection status in this population was young age, where the likelihood of infection decreased with age in rural coyotes but not urban ones. Neither short- nor long-term measures of diet could predict infection across our entire sample, but we found support for our initial hypotheses in young, urban coyotes: both rodent and anthropogenic food consumption effectively predicted E. multilocularis infection in this population. The effects of these predictors were more variable in rural coyotes and older coyotes. We suggest that limiting coyote access to areas in which anthropogenic food and rodent habitat overlap (e.g., compost piles or garbage sites) may effectively reduce the risk of infection, deposition, and transmission of this emerging zoonotic parasite in urban areas.

Detection of Asian genetic components in autochthonous human Echinococcus multilocularis infections from endemic Warmia-Masuria (north-eastern Poland)

Alveolar echinococcosis is a life-threatening zoonotic disease caused by the larval stage of the cestode Echinococcus multilocularis. People are aberrant intermediate hosts accidentally infected with the parasite eggs via faecal-oral route, usually by the consumption of unwashed fruit and vegetable or direct contact with definitive hosts. The recently reported presence of Asian admixture in E. multilocularis tapeworms from Polish red foxes prompted the question of metacestode descent in the human population. In this study, a Maximum Likelihood tree based on partial sequences of E. multilocularis mitochondrial genes cox1, cob, and nad2 coupled with a hierarchical clustering analysis of microsatellite EmsB profiles and supplemented by Sammon's nonlinear mapping with k-means clustering revealed Asian genetic components, to date associated only with the sylvatic cycle, in two autochthonous samples from alveolar echinococcosis patients living in endemic Warmia-Masuria, north-eastern Poland. The red fox is the most likely source of contamination in the environment shared by people and wildlife that led to these infections. Our results confirm that Asian genetic variants participate in the synanthropic cycle in north-eastern Poland and indicate that they may be present in the human population in other areas where Asian genetic variants were detected in red foxes.

Annual assessment of Echinococcus multilocularis surveillance reports submitted in 2023 in the context of commission delegated regulation (EU) 2018/772.

This report is part of the Echinococcus multilocularis surveillance scientific reports which are presented annually by EFSA to the European Commission and are intended to assess the sampling strategy, data collection and detection methods used by Finland, Ireland, United Kingdom (Northern Ireland) and Norway in their respective surveillance programmes. The surveillance programmes of these fourcountries were evaluated by checking the information submitted by each of them and verifying that the technical requirements were fulfilled as laid down in Commission Delegated Regulation (EU) 2018/772 of 21 November 2017 supplementing Regulation (EU) No 576/2013 of the European Parliament and of the Council with regard to preventive health measures for the control of Echinococcus multilocularis infection in dogs, and repealing Delegated Regulation (EU) No 1152/2011. The information was divided into four different categories for assessment: the type and sensitivity of the detection method, the selection of the target population, the sampling strategy and the methodology. For each category, the main aspects that need to be considered in order to accomplish the technical requirements of the legislation were checked against compliance of several criteria. The countries participating in this surveillance (Finland, Ireland, Norway and United Kingdom [Northern Ireland]) succeeded in the fulfilment of the technical legal requirements foreseen in Commission Delegated Regulation (EU) 2018/772 concerning these four different categories None of the four countries recorded positive samples in the 12-month reporting period.

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 duringEchinococcus multilocularisinfection. 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 theE.multilocularisinfection-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 toE.multilocularisinfection. We further showed that mmu-miR-342-3p was able to bind to the 3’ UTR of theZbtb7agene and regulated its expression. Moreover, mmu-miR-342-3p expression was negatively correlated with its target geneZbtb7ain HSCs duringE.multilocularisinfection. Knockdown of mmu-miR-342-3p promoted the expression ofGfapin the activated HSCsin vitro. In theE.multilocularis-infected mice, knockdown of mmu-miR-342-3p suppressed the expression ofα-Sma,Col1α1, andTGF-βbut promoted the expression ofGfap. Therefore, mmu-miR-342-3p is a key regulator for activation of HSCs, and inhibiting mmu-miR-342-3p to suppressed Zbtb7a-mediated TGF-β signaling in activated HSCs could be a novel strategy to treat liver fibrosis induced byE.multilocularis.

Single-cell RNA sequencing deciphers transcriptional profiles of hepatocytes in mouse with hepatic alveolar echinococcosis

To investigate the cell composition and the transcriptional characteristics in microenvironments of hepatic tissues in mice at late stage of Echinococcus multilocularis infection at a single-cell level. Peri-lesion and paired distal hepatic specimens were collected from two BALB/c mice (6 to 8 weeks old) infected with E. multilocularis for single-cell RNA sequencing. The Seurat package in the R software was employed for quality control of data, multi-sample integration and correction of batch effects, and uniform manifold approximation and projection (UMAP) algorithm was used for cell clustering. Cell types were annotated using classical marker genes. Differentially expressed genes were screened in each cell type through differential gene expression analysis, and the biological roles of cells were predicted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. A total of 43 710 cells from peri-lesion and distal hepatic tissues of E. multilocularis-infected mice were analyzed, and were classified into 11 cell types, including neutrophils, T cells, macrophages, granulocyte-monocyte progenitor cells, B cells, plasma cells, basophils, hepatic stellate cells, endothelial cells, hepatocytes, and platelets. T cells were the largest population of immune cells in the microenvironment of hepatic tissues, including five CD4+ T cell subsets, two CD8+ T cell subsets and phosphoantigen-reactive γδT cells. The proportions of CD4+ helper T cells and cytotoxic CD4+ T cells decreased and the proportion of T helper 2 (Th2) cells increased in peri-lesion tissues relative to distal hepatic tissues. In addition, the differentially expressed genes in Th2 cells were associated with negative regulation of the immune system, and the highly expressed genes in cytotoxic CD4+ T cells correlated with activation of the immune system. Single-cell RNA sequencing deciphers the cell composition and distribution in microenvironments of hepatic tissues from mice infected with E. multilocularis, and the increased proportion of Th2 cells in peri-lesion hepatic tissues may be associated with formation of immunosuppressive microenvironments.

Risk Factors and the Character of Clinical Course of the Echinococcus multilocularis Infection in Patients in Poland.

Alveolar echinococcosis (AE) is a chronic zoonotic disease caused by the larval form of Echinococcus multilocularis. In humans, it may become a serious chronic infection of the liver which resembles a slow malignant process leading to death when untreated. The aim of the study was an assessment of the risk factors of the E. multilocularis infections and the description of AE clinical course in the group of 36 patients with confirmed AE, hospitalized at the Department and Clinic of Tropical and Parasitic Diseases, Poznan University of Medical Sciences between 2013 and 2022. Among the study participants, most patients cultivated land, bred livestock, worked in the forest, or were employed in animal shelters. The E. multilocularis infection was diagnosed based on imaging and immunoassay techniques within 6 months in the majority of patients hospitalized in the Department. All patients hospitalized in the Department initiated anti-parasitic therapy at the moment of the diagnosis. Pharmacological treatment combined with surgery was applied in most of the study participants, who were presented with more advanced stages of infection. We conclude the following: 1. For humans in the risk group, regular abdominal imaging examinations and the detection of specific antibodies against E. multilocularis are recommended. 2. Regular screening tests in the hyperendemic areas of AE would increase the early detection of the disease and to improve the clinical prognosis in this extremely life-threatening parasitic disease.

Implementing deep learning models for the classification of Echinococcus multilocularis infection in human liver tissue

BackgroundThe histological diagnosis of alveolar echinococcosis can be challenging. Decision support models based on deep learning (DL) are increasingly used to aid pathologists, but data on the histology of tissue-invasive parasitic infections are missing. The aim of this study was to implement DL methods to classify Echinococcus multilocularis liver lesions and normal liver tissue and assess which regions and structures play the most important role in classification decisions.MethodsWe extracted 15,756 echinococcus tiles from 28 patients using 59 whole slide images (WSI); 11,602 tiles of normal liver parenchyma from 18 patients using 33 WSI served as a control group. Different pretrained model architectures were used with a 60–20–20% random splitting. We visualized the predictions using probability-thresholded heat maps of WSI. The area-under-the-curve (AUC) value and other performance metrics were calculated. The GradCAM method was used to calculate and visualize important spatial features.ResultsThe models achieved a high validation and test set accuracy. The calculated AUC values were 1.0 in all models. Pericystic fibrosis and necrotic areas, as well as germinative and laminated layers of the metacestodes played an important role in decision tasks according to the superimposed GradCAM heatmaps.ConclusionDeep learning models achieved a high predictive performance in classifying E. multilocularis liver lesions. A possible next step could be to validate the model using other datasets and test it against other pathologic entities as well, such as, for example, Echinococcus granulosus infection.Graphical

A multi-epitope vaccine GILE against Echinococcus Multilocularis infection in mice.

The objective of this study is to construct a multi-epitope vaccine GILE containing B-cell and T-cell epitopes against Echinococcus Multilocularis (E. multilocularis) infection based on the dominant epitopes of E. multilocularis EMY162, LAP, and GLUT1. The structure and hydrophobicity of GILE were predicted by SWISSMODEL, pyMOL, SOPMA and VMD, and its sequence was optimized by Optimum™ Codon. The GILE gene was inserted into pCzn1 and transformed into Escherichia coli Arctic express competent cells. IPTG was added to induce the expression of recombinant proteins. High-purity GILE recombinant protein was obtained by Ni-NTA Resin. BALB/c mice were immunized with GILE mixed with Freund's adjuvant, and the antibody levels and dynamic changes in the serum were detected by ELISA. Lymphocyte proliferation was detected by MTS. The levels of IFN-g and IL-4 were detected by ELISpot and flow cytometry (FCM). T cells were detected by FCM. The growth of hepatic cysts was evaluated by Ultrasound and their weights were measured to evaluate the immune protective effect of GILE. The SWISS-MODEL analysis showed that the optimal model was EMY162 95-104-LAP464-479-LAP495-510-LAP396-410-LAP504-518-EMY162112-126. The SOPMA results showed that there were Alpha helix (14.88%), Extended strand (26.25%), Beta turn (3.73%) and Random coil (45.82%) in the secondary structure of GILE. The restriction enzyme digestion and sequencing results suggested that the plasmid pCzn1-GILE was successfully constructed. The SDSPAGE results indicated that the recombinant protein was 44.68 KD. The ELISA results indicated that mice immunized with GILE showed higher levels of serum antibodies compared to the PBS group. The FCM and ELISpot results indicated that mice immunized with GILE secreted more IFN-g and IL-4. Immunization with GILE also led to a significant decrease in the maximum diameter and weight of cysts and stimulated the production of CD4+ and CD8+ T Cell. A multi-epitope vaccine GILE with good immunogenicity and antigenicity has been successfully constructed in this study, which may provide important theoretical and experimental bases for the prevention and treatment of E. multilocularis infection.

A survey of gastrointestinal parasites in dogs illegally entering the UK (2015-2017).

This study involving non-compliant, seized dogs entering the UK surveyed endoparasites detected in faecal samples. A focus was placed on taeniid infection as the detection of these tapeworms acts as a marker for failure of effective tapeworm treatment. Individual faecal samples taken from 65 dogs over a 24-month period were examined for helminth eggs, for protozoal oocysts and cysts, using a centrifugal flotation technique. Any sample presenting positive results for taeniid eggs had residual faeces examined using polymerase chain reaction to aid speciation of the tapeworm eggs. Additionally, a Baermann technique was used to assess faeces for lungworm larvae. Patent endoparasite infection was detected in 27.7% of dog faecal samples. No sample was positive for lungworm larvae. Five dogs were co-infected with Isospora spp. and Toxocara canis. One dog sample was detected with taeniid eggs, identified as Taenia serialis. The taeniid-positive dog indicated that appropriate tapeworm treatment may not have occurred, reinforcing the risk to the UK of illegally imported dogs potentially introducing Echinococcus multilocularis infection.

Differential Activity of Human Leukocyte Extract on Systemic Immune Response and Cyst Growth in Mice with Echinococcus Multilocularis Infection After Oral, Subcutaneous and Intraperitoneal Routes of Administration.

Alveolar echinococcosis (AE) caused by the larval stage of Echinococcus multilocularis is serious parasitic diseases associated with the host´s immunosuppression. The effects of human non-immune dialyzable leukocyte extract (DLE) on immune cells in blood and spleen and parasitic cysts weight in Balb/c mice after oral (PO), subcutaneous (SC) and intraperitoneal administration (IP) were compared. The reduction in cysts weight (p < 0.01) was recorded after PO route, whereas moderate reduction was found after SC and IP routes. The elevation of lymphoid populations in blood and spleen was found after PO administration (p < 0.01) in parallel with reduced myeloid population. Infection-elicited decline in B220+B cells was partially abolished by PO route, but DLE routes did not influence the CD3+ T cells. The proportions of CD3+CD4+Th lymphocytes were moderately upregulated, whereas CD3+CD8+Tc populations were reduced after all DLE routes (p < 0.01). PO administration increased CD11b+MHCIIhigh blood monocytes, CD11b-SigleF+ cell, but not CD11b+Si-glecF+ eosinophils in the blood, stimulated after SC and IP routes. DLE induced downregulation of NO production by LPS-stimulated adherent splenocytes ex vivo. Con A-triggered T lymphocyte proliferation was associated with the elevated IFN-γ production and transcription factor Tbet mRNA expression. The alleviation of Th2 (IL-4) and Treg (TGF-β) cytokine production by lymphocytes ex vivo paralleled with downregulation of gene transcription for cytokines, GATA and FoxP3. Reduction of myeloid cells with suppressive activity was found. The SC and IP routes affected partially the cysts weights, diminished significantly gene transcription, NO levels and Th2 and Treg cytokines production. Results showed that PO route of DLE administration was the most effective in ameliorating immunosuppression via stimulation of Th1 type, reducing Th2 and Treg type of immunity and CD3+CD8+Tc lymphocytes in the blood and spleens during E. multilocularis infection in mice.