An in vivo appraisal of Punica granatum peel extract's ultrastructural effect on cystic echinococcosis in mice.

In the past decade, interest has significantly increased regarding the medicinal and nutritional benefits of pomegranate (Punica granatum) peel. This study examined the effects of using pomegranate peel extract (PGE) alone and in combination with albendazole (ABZ) on ultrastructural and immunological changes in cystic echinococcosis in laboratory-infected mice. Results revealed that the smallest hydatid cyst size and weight (0.48 ± 0.47mm, 0.17 ± 0.18 gm) with the highest drug efficacy (56.2%) was detected in the PGE + ABZ group, which also exhibited marked histopathological improvement. Ultrastructural changes recorded by transmission electron microscopy including fragmentation of the nucleus, glycogen depletion, and multiple lysosomes in vacuolated cytoplasm were more often observed in PGE + ABZ group. IFN-γ levels were significantly increased in the group treated with ABZ, with a notable reduction following PGE treatment, whether administered alone or in combination with ABZ. Thus, PGE enhanced the therapeutic efficiency of ABZ, with improvement in histopathological and ultrastructural changes.

Echinococcus granulosus ubiquitin-conjugating enzymes (E2D2 and E2N) promote the formation of liver fibrosis in TGFß1-induced LX-2 cells.

BACKGROUND: Cystic echinococcosis (CE) is a widespread zoonosis caused by the infection with Echinococcus granulosus sensu lato (E. granulosus s.l.). CE cysts mainly develop in the liver of intermediate hosts, characterized by the fibrotic tissue that separates host organ from parasite. However, precise mechanism underlying the formation of fibrotic tissue in CE remains unclear. METHODS: To investigate the potential impact of ubiquitin-conjugating enzymes on liver fibrosis formation in CE, two members of ubiquitin-conjugating (UBC) enzyme of Echinococcus granulosus (EgE2D2 and EgE2N) were recombinantly expressed in Escherichia coli and analyzed for bioinformatics, immunogenicity, localization, and enzyme activity. In addition, the secretory pathway and their effects on the formation of liver fibrosis were also explored. RESULTS: Both rEgE2D2 and rEgE2N possess intact UBC domains and active sites, exhibiting classical ubiquitin binding activity and strong immunoreactivity. Additionally, EgE2D2 and EgE2N were widely distributed in protoscoleces and germinal layer, with differences observed in their distribution in 25-day strobilated worms. Further, these two enzymes were secreted to the hydatid fluid and CE-infected sheep liver tissues via a non-classical secretory pathway. Notably, TGFß1-induced LX-2 cells exposed to rEgE2D2 and rEgE2N resulted in increasing expression of fibrosis-related genes, enhancing cell proliferation, and facilitating cell migration. CONCLUSIONS: Our findings suggest that EgE2D2 and EgE2N could secrete into the liver and may interact with hepatic stellate cells, thereby promoting the formation of liver fibrosis.

Baiting not-owned dogs against Echinococcus granulosus: innovative tools for integrated control.

Cystic echinococcosis (CE), caused by the larval stage of the cestode Echinococcus granulosus, is one of the most widespread zoonoses in Mediterranean countries. Baiting not-owned dogs with praziquantel (PZQ), due to their key role in the maintaining the transmission of CE, currently appears to be the most effective way to limit the transmission of CE, as well as an important aspect to introduce for the control of this parasitic disease. Therefore, this study aims to test 3 types of PZQ-based baits by evaluating different parameters (integrity over time, attractiveness and palatability for dogs, and mechanical resistance after release to different altitudes) and the bait acceptance in field by target animals, i.e. not-owned dogs, by using camera traps. The double PZQ-laced baits (with a double layer of highly palatable chews) showed the greatest resistance in the environment while also preserving the attractiveness and palatability up to 10 days, also withstood heights of 25 m, thus resulting as the most suitable also for drone delivery. The results on the field showed that most of the baits were consumed by not-owned dogs (82.2%), while the remaining were consumed by wild boars (8.9%), foxes (6.7%), badgers (1.1%) and hedgehogs (1.1%), confirming the specific and high attractiveness of the double PZQ-laced baits for the target population and highlights how an anthelmintic baiting programme may be a viable tool for the management of E. granulosus among free-ranging dog populations in endemic rural areas.

Echinococcus granulosus comparative genotyping in sheep in Saudi Arabia and Egypt.

Background: Cystic echinococcosis (CE), which is triggered by the parasite Echinococcus granulosus, is a global zoonotic disease that is common in rural regions in which there are frequent encounters between dogs and other domestic animals. The disease can have devastating consequences, impacting the health of people and animals and leading to huge financial losses, especially in the agricultural industry. In the Kingdom of Saudi Arabia (KSA) and Egypt, despite the high incidence of disease, few investigations have been conducted into the genetic variation in species of the genus Echinococcus. Aim: This study sought to compare the genetic features of the hydatid cysts carried in sheep in KSA with those found in Egypt. Methods: DNA from the protoscolices was used in a PCR targeting the mitochondrial NADH dehydrogenase 1 (NAD1), cytochrome c oxidase subunit 1 (COX1), and nuclear actin II (ACT II) genes, and the resulting amplification products of 30 KSA and Egyptian isolates were sequenced and compared. Results: Among the sheep in KSA, the overall prevalence of CE was 0.51%. Of the sheep cyst DNA samples, 95%, 100%, and 52% were positive for the Cox1, nad1, and act II genes, respectively. Targeting all three genes, all KSA samples belonged to the E. granulosus genotype (G1), whereas all Egyptian isolates belonged to E. granulosus (G1) and E. canadensis (G6). Conclusion: We conclude that isolates of E. granulosus from the two countries shared a common origin in Arabic North Africa, with sheep and camels as common hosts.

Gastrointestinal parasites of dogs and foxes in the Zanjan province of Iran: With an emphasis on Echinococcus species.

Canids harbor many zoonotic parasites and play an important role in the spread of parasites in the human environment. Estimation of parasitic infection among canids as definitive hosts may help competent authorities design efficient control programs. This study was conducted to determine the prevalence of intestinal parasites in dogs and foxes with an emphasis on Echinococcus spp. A total of 500 fecal samples of dogs and 30 fecal samples of foxes were studied in the summer, autumn, and winter of 2021 in the Zanjan province using the formalin-ethyl acetate concentration technique, followed by multiplex PCR. The overall prevalence of gastrointestinal parasite infection was estimated to be 19.05%. The prevalence was 24.8%, 10.2%, and 26.7% in stray, shelter dogs and foxes, respectively. No parasites were found among pet and guard dog samples. PCR results on Taenidae eggs showed that 2.4% of samples were positive for Echinococcus granulosus and none contained E. multilocularis. Noteworthy is that E. granulosus was identified only in stray dog samples. The higher prevalence of E. granulosus infection in stray dogs in this province emphasizes the importance of monitoring the food sources consumed by these animals.

Untargeted metabolomics to discriminate liver and lung hydatid cysts: Importance of metabolites involved in the immune response.

The Echinococcus granulosus sensu lato species complex is responsible for the neglected zoonotic disease known as cystic echinococcosis (CE). Humans and livestock are infected via fecal-oral transmission. CE remains prevalent in Western China, Central Asia, South America, Eastern Africa, and the Mediterranean. Approximately one million individuals worldwide are affected, influencing veterinary and public health, as well as social and economic matters. The infection causes slow-growing cysts, predominantly in the liver and lungs, but can also develop in other organs. The exact progression of these cysts is uncertain. This study aimed to understand the survival mechanisms of liver and lung CE cysts from cattle by determining their metabolite profiles through metabolomics and multivariate statistical analyses. Non-targeted metabolomic approaches were conducted using quadrupole-time-of-flight liquid chromatography/mass spectrometry (LC-QTOF-MS) to distinguish between liver and lung CE cysts. Data processing to extract the peaks on complex chromatograms was performed using XCMS. PCA and OPLS-DA plots obtained through multiple statistical analyses showed interactions of metabolites within and between groups. Metabolites such as glutathione, prostaglandin, folic acid, and cortisol that cause different immunological reactions have been identified both in liver and lung hydatid cysts, but in different ratios. Considering the differences in the metabolomic profiles of the liver and lung cysts determined in the present study will contribute research to enlighten the nature of the cyst and develop specific therapeutic strategies.

Comparison of the Scolicidal Activity of Two Leaves Extracts of Myrtus communis from Algeria Against Echinococcus granulosus Sensu Lato Protoscoleces.

PURPOSE: During cystic echinococcosis surgery, the use of scolicidal agents such as hypertonic saline (20%) aims to reduce the risk of infection recurrence, but most of the used agents are associated with undesirable side effects. Therefore, the use of natural scolicidal agents such as medicinal plant extracts could reduce these medical issues. The present study aimed to compare in vitro the scolicidal activity between two extracts of the medicinal plant Myrtus communis from Algeria against Echinococcus granulosus sensu lato protoscoleces. METHODS: The ethanolic and aqueous extraction of plant leaves was performed. Phytochemical analysis by gas chromatography-tandem mass spectrometry (GC-MS/MS), determination of total phenolic and flavonoid contents, and in vitro antioxidant activity by DPPH were evaluated for both extracts. Finally, the in vitro scolicidal activity was tested by different concentrations. The viability was evaluated by the eosin exclusion test. RESULTS: The phytochemical analysis revealed 28 components for the ethanolic extract and 44 components for the aqueous extract. The major components were 2'-hydroxy-5'-methoxyacetophenone and 4-amino-2-methylphenol, respectively. The total phenolic and flavonoid contents were 45.9 ± 0.085 mg of gallic acid equivalent per g of extract (GAE/g E) and 16.5 ± 0.004 mg of quercetin equivalent per g (QE/g E) for the ethanolic extract, and 36.5 ± 0.016 mg GAE/g E and 18.2 ± 0.023 mg QE/g E for the aqueous extract, respectively. Furthermore, ethanolic and aqueous extracts of M. communis gave a value of IC50 = 0.009 ± 0.0004 mg/ml and IC50 = 0.012 ± 0.0003 mg/ml for the antioxidant activity, respectively. The in vitro scolicidal activity with concentrations of 50, 75, 100, and 150 mg/ml was tested for 5, 10, 15, and 30 min, and 5, 10, 15, 30, 60, 90, and 120 min for ethanolic and aqueous extracts, respectively. The mortality rate of protoscoleces at concentrations of 100 and 150 mg/ml was 98.8 and 100%, respectively, after 5 min of exposure to the ethanolic extract, while this rate was 100% at the same concentrations only after 60 min of exposure to the aqueous extract. CONCLUSIONS: The ethanolic extract showed a stronger scolicidal activity against E. granulosus s.l protoscoleces than the aqueous extract. In the future, other investigations are necessary to elucidate the mechanism of action and the possible toxicity on human cells. Moreover, experimental animal studies are required to investigate the efficacy of different extracts of this plant and its components as natural anti-parasitic alternatives for the treatment of human cystic echinococcosis.

Mouse model of secondary cystic echinococcosis.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of the cestode Echinococcus granulosus sensu lato (s. l.), a genetic complex composed of five species: E. granulosus sensu stricto (s. s.), E. equinus, E. ortleppi, E. canadensis, and E. felidis. The parasite requires two mammalian hosts to complete its life cycle: a definitive host (mainly dogs) harboring the adult parasite in its intestines, and an intermediate host (mostly farm and wild ungulates) where hydatid cysts develop mainly in the liver and lungs. Humans are accidental intermediate hosts, being susceptible to either primary or secondary forms of CE; the first one due to the ingestion of oncospheres, and the second one because of the spillage of protoscoleces (PSC) contained within a primary cyst. Secondary CE is a serious medical problem, and can be modeled in immunocompetent mice (a non-natural intermediate host) through the intraperitoneal inoculation of viable PSC from E. granulosus s. l. This model is useful to study not only the immunobiology of CE, but also to test new chemotherapeutics or therapeutical protocols, to explore novel vaccine candidates, and to evaluate alternative diagnostic and/or follow-up tools. The mouse model of secondary CE involves two sequential stages: an early stage of parasite pre-encystment (PSC develop into hydatid cysts in the peritoneal cavity of mice), and a late or chronic stage of parasite post-encystment (already differentiated cysts slowly grow during the whole host lifespan). This model is a time-consuming infection, whose outcome depends on several factors like the parasite infective dose, the mouse strain, and the parasite species/genotype. Thus, such variables should always be adjusted according to the research objectives. Herein, the general materials and procedures needed to establish secondary CE in mice are described, as well as several useful tips and recommendations.

Effects of annexin B18 from Echinococcus granulosus sensu lato on mouse macrophages.

Cystic echinococcosis (CE) is a zoonotic disease, caused by Echinococcus granulosus sensu lato (E. granulosus s. l.), which posed significant public health concern globally. E. granulosus s. l. annexin B18 (EgANXB18) acts as a secretory protein, exerting a crucial influence in mediating host-parasite interactions. Recombinant annexin B18 (rEgANXB18) was expressed by Escherichia coli and the immunoreactivity was assessed by western blotting. The binding affinity between rEgANXB18 and total protein of RAW264.7 cells was assessed by ELISA. The impact of rEgANXB18 on the metabolic activity of RAW264.7 cells was assayed by Cell Counting Kit-8 assay. The mRNA levels of polarization markers (inducible nitrous oxide synthase (iNOS) and arginase 1 (Arg1)) and key cellular factors (IL-1ß，IL-6，IL-10 and TNFα) were evaluated by qRT-PCR. rEgANXB18 was successfully expressed and recognized by E. granulosus s.l. infected canine sera, as well as could bind to the total protein of RAW264.7 cells. Additionally, rEgANXB18 could promote metabolic activity at 5, 10, 20, and 40 µg/mL while no significant impact on metabolic activity was observed at 80 µg/mL. Co-culture RAW264.7 cells with rEgANXB18 resulted in significantly upregulation of the transcript levels of polarization markers iNOS and Arg1. Moreover, rEgANXB18 significantly upregulated the transcript levels of IL-1ß, IL-6, TNFα, and IL-10, while dose-effect relationship was observed in IL-1ß, IL-6, and IL-10. Our results indicated that EgANXB18 showed the potential to regulate immune response of macrophages by shifting the cell polarization and cytokine profile, thereby promoting the parasitism of CE.

Molecular analyses of exosome-derived miRNAs revealed reduced expression of miR-184-3p and decreased exosome concentration in patients with alveolar echinococcosis.

Both E. multilocularis and host-derived exosomes are involved in the pathogenic process of alveolar echinococcosis (AE). Exosomes secrete miRNAs that have regulatory roles in host-pathogen interactions in multiple ways. In the present study, we collected and purified supernatants of E. multilocularis cultures, as well as human plasma exosomes. High-throughput sequencing showed the identities of 45 exosomal miRNAs in E. multilocularis. The lengths of these miRNAs ranged from 19 to 25 nucleotides (nt), with the majority (n = 18) measuring 22 nt. Notably, emu-let-7-5p emerged as the most abundant among these miRNAs, with a detected count of 33,097 and also length of 22 nt. Nanoparticle tracking analysis (NTA) showed that the concentration of exosomes in the plasma of AE patients was lower compared to that in the healthy individuals. This result suggested that the concentration of plasma exosomes was able to distinguish AE patients from healthy individuals. Using qRT-PCR to assess the relative expression of 10 miRNAs of E. multilocularis, we showed that the expression of miR-184-3p was downregulated significantly in the exosomes of plasma from AE patients compared to that in the control group. In summary, this study indicates that AE induces a reduction in the concentration of human plasma exosomes, as well as downregulating miR-184-3p in infected individuals.

Weighted gene co-expression network analysis reveals immune evasion related genes in Echinococcus granulosus sensu stricto.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

Assessing the Potential Apoptotic Effects of Different Hydatid Cyst Fluids on Human Healthy Hepatocytes and Hepatocellular Carcinoma Cells.

Cystic Echinococcosis (CE) is a zoonotic infection caused by the larval form of Echinococcus granulosus in humans. Emerging evidence suggests an intriguing inverse association between E. granulosus infection and the occurrence of cancer. This study aimed to investigate the influence of diverse host-derived hydatid cyst fluids (HCF) with distinct genotypes on human liver hepatocytes (HC) and hepatocellular carcinoma cells (HepG2). Specifically, we examined their effects on cell proliferation, apoptosis sensitivity (BAX/BCL-2), apoptosis-related p53 expression, and the expression of cancer-related microRNA (hsa-miR-181b-3p). Cell proliferation assays, real-time PCR, and ELISA studies were conducted to evaluate potential anti-cancer properties. The findings revealed that animal-origin HCF (G1(A)) induced direct cell death by augmenting the susceptibility of HepG2 cells to apoptosis. Treatment with both G1(A) and G1(H) HCF sensitized HepG2 and HC cell lines to apoptosis by modulating the BAX/BCL-2 ratio, accompanied by upregulation of the p53 gene. Additionally, G1(A) HCF and human-derived HCFs (G1(H), G7(H)) reduced the expression of miR-181b-3p in HepG2 cells. Consequently, this study demonstrates the potential anti-cancer effect of HCF in HepG2 cells and provides the first comparative assessment of HCFs from human and animal sources with diverse genotypes, offering novel insights into this field.

Inhibition of AMPK activation in Echinococcus granulosus sensu stricto limits the parasite's glucose metabolism and survival.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by larvae of the Echinococcus granulosus sensu lato (s.l.) cluster. There is an urgent need to develop new drug targets and drug molecules to treat CE. Adenosine monophosphate (AMP)-activated protein kinase (AMPK), a serine/threonine protein kinase consisting of α, ß, and γ subunits, plays a key role in the regulation of energy metabolism. However, the role of AMPK in regulating glucose metabolism in E. granulosus s.l. and its effects on parasite viability is unknown. In this study, we found that targeted knockdown of EgAMPKα or a small-molecule AMPK inhibitor inhibited the viability of E. granulosus sensu stricto (s.s.) and disrupted the ultrastructure. The results of in vivo experiments showed that the AMPK inhibitor had a significant therapeutic effect on E. granulosus s.s.-infected mice and resulted in the loss of cellular structures of the germinal layer. In addition, the inhibition of the EgAMPK/EgGLUT1 pathway limited glucose uptake and glucose metabolism functions in E. granulosus s.s.. Overall, our results suggest that EgAMPK can be a potential drug target for CE and that inhibition of EgAMPK activation is an effective strategy for the treatment of disease.

Assessment of oxidative stress and tissue damage in Echinococcus granulosus naturally infected bovine liver.

Echinococcus granulosus is a zoonotic parasite infects many livestock species, especially cattle, sheep, goat and buffalo, causing cystic echinococcosis. The aim of this study was to demonstrate the presence of the parasite and parasitic tissue damage histopathologically and to determine the role of oxidative stress in the tissue damage through the immunohistochemical detection of the oxidative damage-marker malondialdehyde (MDA) and the antioxidant response-marker superoxide dismutase (SOD). The material of the study consisted of 20 liver samples with Echinococcus cysts and 10 E.granulosus- negative healthy liver samples obtained from different cattle at various times from slaughterhouses in Kirikkale province, Turkey. Histopathologically, Echinococcus cysts of various sizes were observed along with the surrounding fibrous connective tissue. Giant cells, mononuclear cells, and eosinophilic leukocytes were found between the fibrous connective tissue and the cyst. In the parenchymal tissue distant from the cyst, inflammatory changes were observed, including vacuolation and necrosis in hepatocytes, congestion and dilation sinusoidal capillaries. Immunohistochemically, MDA immunopositivity was observed in both hepatocytes surrounding the cyst and areas distant from the cyst, while SOD immunopositivity was mainly detected in fibrous connective tissue and hepatocytes surrounding the Echinococcus cysts. A significant increase in MDA immunoreactivity was observed in E.granulosus s.l.-infected livers. Although no statistically significant change was observed in SOD immunopositivity in the liver tissues with cystic echinococcosis, regional variations were noted. Germinal layer (GL) of Echinococcus cyst showed immunopositive staining for MDA, while laminated layer (LL) exhibited immunonegative staining. To the authors' best understanding, this study represents a pioneering effort in showcasing and evaluating the immunoreactivities of MDA and SOD within the liver tissue afflicted with Echinococcus cysts. Simultaneously, the examination extends to encompass tissue damage and the infiltration of inflammatory cells. This study highlights the role of oxidative stress in the pathogenesis of Cystic Echinococcosis (CE) and the need for further investigation of antioxidant defense mechanisms and their regional variations.

Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

Human alveolar echinococcosis in Slovakia: Epidemiology and genetic diversity of Echinococcus multilocularis, 2000-2023.

Human alveolar echinococcosis (AE) is a serious parasitic disease caused by larval stages of Echinococcus multilocularis. Between January 2000 and October 2023, 137 AE cases were confirmed in Slovakia. The average annual incidence increased from 0.031 per 100,000 inhabitants between 2000 and 2011, to an average of 0.187 since 2012, i.e. about six times. Among patients, 45.3% were men and 54.7% were women; the mean age at the time of diagnosis was 52.8 years. Most cases were diagnosed in the age groups 51-60 years and 61-70 years (33 cases each), and eight patients fell into the age category ≤ 20 years. To better recognize the gene diversity in clinical samples, metacestodes from 21 patients collected between 2013 and 2021 were subjected to DNA sequencing of four mitochondrial genes. Using concatenated sequences of cob (603 bp), nad2 (882 bp) and cox1 (789 bp) gene fragments, 14 isolates (66.7%) were assigned to the European E5 profile of E. multilocularis, two isolates (9.5%) to the E5a subtype, four isolates (19%) to the E4 profile, and one isolate (4.8%) to haplogroup E1/E2. The E5-type profiles and E4 profiles were distributed throughout the country, whereas the E1/E2 profile was found in the patient from western Slovakia. According to the data obtained and GenBank sequences, the E5-type dispersal is so far limited to central-eastern Europe and the variant seems to be indigenous to that region. The admixture with the haplotypes E4 and E1/E2 could have taken place from a historical endemic focus during the fox expansion in the last decades. By employing the nad1 fragment, a typical European haplotype was observed in all 21 resolved Slovak samples. The acceleration in the AE incidence in the last decade suggests the emergence of the disease and the need for further research on human and animal isolates.

Viability predictive factors of the daughter vesicles in hepatic cystic echinococcosis.

INTRODUCTION: Management of cystic echinococcosis (CE) requires knowledge of certain aspects related to the survival of Echinococcus granulosus. The viability of daughter vesicles (DV) is a determining factor in guiding therapeutic indications, particularly for transiently active Cysts type CE3b. PURPOSE: To determine the predictive factors of DV viability and its impact on the therapeutic management of CE3b type. MATERIALS AND METHODS: This is a prospective pilot study with an analytical aim on patients with cystic echinococcosis of the liver type CE2 and CE3b, operated in the General Surgery Department of Habib-Bourguiba Academic Hospital, Sfax-Tunisia for 22 months from March 2018 until December 2019. The unit of the study is the DV. A parasitological study of the DV was done in the parasitology laboratory. RESULTS: During the study period, 27 (40.9%) of 66 operated CE Disease from 21 patients containing 248 DV were explored. The median viability of DV protoscoleces was 16.7%. In bivariate analysis, factors for viability of DV protoscoleces were: fever, acute cholangitis, hyperbilirubinemia, left liver location, rock water and bilious echinococcal fluid (EF), cyst size ≥ 43 mm, Intracystic pressure ≥ 35 mmHg, DV size ≥ 6.5 mm, volume, number of DV/cyst ≥ 5, and opaque wall (p < 0.05). Predictive factors for the Non-viability of DV were: CE3b type, purulent EF, gelatinous EF. In multivariate analysis, only CE2 type, cyst size ≥ 43 mm, number of DV/cyst ≥ 5 and DV size ≥ 6.5 mm were factors significantly associated with the viability of DV protoscoleces. CONCLUSION: CE3b cysts without the criteria of viability of DV protoscoleces may become candidates for the 'Wait-and-Watch' procedure.

Effects of protoscoleces excretory-secretory products of Echinococcus granulosus on hepatocyte growth, function, and glucose metabolism.

Cystic echinococcosis (CE) is one of the most widespread and harmful zoonotic parasitic diseases, which most commonly affects the liver. In this study, we characterized multiple changes in mouse hepatocytes following treatment with excretory-secretory products (ESPs) of Echinococcus granulosus protoscoleces (Eg-PSCs) by a factorial experiment. The cell counting kit-8 assay (CCK-8), the 5-ethynyl-2'-deoxyuridine (EdU) assay, and flow cytometry were used to detect the growth of hepatocytes. Inverted microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to observe the morphology and ultrastructure of hepatocytes. An automatic biochemical analyzer and an ELISA detection kit were used to determine six conventional hepatocyte enzymatic indices, the levels of five hepatocyte-synthesized substances, and the contents of glucose and lactate. Western blot analysis was conducted to analyze the protein expression of three apoptosis-related proteins, Bax, Bcl-2, cleaved caspase-3, and six glucose metabolism pathways rate-limiting enzymes in hepatocytes. The results showed that ESPs inhibited hepatocyte proliferation and promoted hepatocyte apoptosis. The cell membrane and microvilli of hepatocytes changed, and the nucleus, mitochondria and rough endoplasmic reticulum were damaged to varying degrees. The contents of iron, albumin (ALB), uric acid (UA) and urea were increased, and the activities of six enzymes in hepatocytes were increased except for the decrease of transferrin (TRF). The expression levels of all six key enzymes in the glucose metabolism pathway in hepatocytes were reduced. Our characterization provides a basis for further research on the pathogenesis, prevention and treatment of CE.

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 (ß = 0.391, p = 0.006). In contrast, on the local scale, the mean annual precipitation (ß = 0.285, p = 0.008) and the precipitation seasonality (ß = - 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.

Comparative characterization of microRNA-71 of Echinococcus granulosus exosomes.

Cystic echinococcosis (CE) is a global zoonotic disease caused by Echinococcus granulosus, posing a great threat to human and animal health. MiRNAs are small regulatory noncoding RNA involved in the pathogenesis of parasitic diseases, possibly via exosomes. Egr-miR-71 has been identified as one of the miRNAs in the blood of CE patients, but its secretory characteristics and functions remains unclear. Herein, we studied the secretory and biological activity of exosomal egr-miR-71 and its immunoregulatory functions in sheep peripheral blood mononuclear cells (PBMCs). Our results showed that egr-miR-71 was enriched in the exosome secreted by protoscoleces with biological activity. These egr-miR-71-containing exosomes were easily internalized and then induced the dysregulation of cytokines (IL-10 and TNF-α), nitric oxide (NO) and key components (CD14 and IRF5) in the LPS/TLR4 pathway in the coincubated sheep PBMCs. Similarly, egr-miR-71 overexpression also altered the immune functions but exhibited obvious differences in regulation of the cytokines and key components, preferably inhibiting proinflammatory cytokines (IL-1α, IL-1ß and TNF-α). These results demonstrate that exosomal egr-miR-71 is bioactive and capacity of immunomodulation of PBMCs, potentially being involved in immune responses during E. granulosus infection.

Title: Caractérisation comparative du microARN-71 des exosomes d'Echinococcus granulosus. Abstract: L'échinococcose kystique (EK) est une maladie zoonotique mondiale causée par Echinococcus granulosus, représentant une grande menace pour la santé humaine et animale. Les miARN sont des petits ARN régulateurs non codants impliqués dans la pathogenèse des maladies parasitaires, éventuellement via les exosomes. Egr-miR-71 a été identifié comme l'un des miARN présents dans le sang des patients atteints d'EK, mais ses caractéristiques et fonctions sécrétoires restent floues. Ici, nous avons étudié l'activité sécrétoire et biologique du egr-miR-71 exosomal et ses fonctions immunorégulatrices dans les cellules mononucléées du sang périphérique (CMSP) de mouton. Nos résultats ont montré qu'egr-miR-71 était enrichi dans l'exosome sécrété par les protoscolex ayant une activité biologique. Ces exosomes contenant egr-miR-71 ont été facilement internalisés et ont ensuite induit la dérégulation des cytokines (IL-10 et TNF-α), de l'oxyde nitrique (NO) et des composants clés (CD14 et IRF5) de la voie LPS/TLR4 dans les CMSP de mouton co-incubées. De même, la surexpression d'egr-miR-71 a également modifié les fonctions immunitaires mais a montré des différences évidentes dans la régulation des cytokines et des composants clés, inhibant de préférence les cytokines pro-inflammatoires (IL-1α, IL-1ß et TNF-α). Ces résultats démontrent que l'egr-miR-71 exosomal est bioactif et possède une capacité d'immunomodulation des CMSP, potentiellement impliquée dans les réponses immunitaires lors d'une infection à E. granulosus.