Human alveolar echinococcosis is a hard-to-treat and largely untreated parasitic disease with high associated health care costs. The current antiparasitic treatment for alveolar echinococcosis relies exclusively on albendazole, which does not act parasiticidally and can induce severe adverse effects. Alternative, and most importantly, improved treatment options are urgently required. A drug repurposing strategy identified the approved antimalarial pyronaridine as a promising candidate against Echinococcus multilocularis infections. Following a 30-day oral regimen (80 mg kg−1 day−1), pyronaridine achieved an excellent therapeutic outcome in a clinically relevant hepatic alveolar echinococcosis murine model, showing a significant reduction in both metacestode size (72.0%) and counts (85.2%) compared to unmedicated infected mice, which revealed significantly more potent anti-echinococcal potency than albendazole treatment at an equal dose (metacestode size: 42.3%; counts: 4.1%). The strong parasiticidal activity of pyronaridine was further confirmed by the destructive damage to metacestode tissues observed morphologically. In addition, a screening campaign combined with computational similarity searching against an approved drug library led to the identification of pirenzepine, a gastric acid-inhibiting drug, exhibiting potent parasiticidal activity against protoscoleces and in vitro cultured small cysts, which warranted further in vivo investigation as a promising anti-echinococcal lead compound. Pyronaridine has a known drug profile and a long track record of safety, and its repurposing could translate rapidly to clinical use for human patients with alveolar echinococcosis as an alternative or salvage treatment.

Cystic and alveolar echinococcosis are considered the second and third most significant foodborne parasitic diseases worldwide. The microscopic eggs excreted in the feces of the definitive host are the only source of contamination for intermediate and dead-end hosts, including humans. However, estimating the respective contribution of the environment, fomites, animals or food in the transmission of Echinococcus eggs is still challenging. Echinococcus granulosus and E. multilocularis seem to have a similar survival capacity regarding temperature under laboratory conditions. In addition, field experiments have reported that the eggs can survive several weeks to years outdoors, with confirmation of the relative susceptibility of Echinococcus eggs to desiccation. Bad weather (such as rain and wind), invertebrates and birds help scatter Echinococcus eggs in the environment and may thus impact human exposure. Contamination of food and the environment by taeniid eggs has been the subject of renewed interest in the past decade. Various matrices from endemic regions have been found to be contaminated by Echinococcus eggs. These include water, soil, vegetables and berries, with heterogeneous rates highlighting the need to acquire more robust data so as to obtain an accurate assessment of the risk of human infection. In this context, it is essential to use efficient methods of detection and to develop methods for evaluating the viability of eggs in the environment and food.

Manipulation of host behaviour by parasites to enhance transmission to the next host is a fascinating phenomenon that has interested scientists since the 1970s. It has been proposed that infection with the cestode Echinococcus multilocularis produces an impairment of the antipredatory behaviour in the rodent intermediate host common vole, Microtus arvalis, which may facilitate transmission of the tapeworm to the canid final host. In this study, we observed the behaviour of infected common voles at 12 weeks post-infection, when protoscoleces production and maturation commonly occurs, in order to assess behavioural changes compared to uninfected controls, that might ease predation in the wild. Infected and uninfected voles were monitored for 24 h to observe their spontaneous activity. In addition, the next day, both infected and uninfected voles were subjected to 4 different behavioural tests: open field test, barrier test, platform test and air-puff test in a running wheel. No significant difference between uninfected and infected voles emerged during the behavioural tests. However, observation of spontaneous activity revealed that infected voles increased their feeding frequency and spent significantly more time above bedding even when not eating, compared to the uninfected controls. In the wild, these behavioural changes increase the animals exposure to predators, raising their chance of becoming prey. These findings are the first direct evidence consistent with behavioural manipulation by E. multilocularis on common voles.

Echinococcus canadensis consists of 4 genotypes: G6, G7, G8 and G10. While the first 2 predominantly infect domestic animals, the latter are sylvatic in nature involving mainly wolves and cervids as hosts and can be found in the northern temperate to Arctic latitudes. This circumstance makes the acquisition of sample material difficult, and little information is known about their genetic structure. The majority of specimens analysed to date have been from the European region, comparatively few from northeast Asia and Alaska. In the current study, Echinococcus spp. from wolves and intermediate hosts from the Republic of Sakha in eastern Russia were examined. Echinococcus canadensis G10 was identified in 15 wolves and 4 cervid intermediate hosts. Complete mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences were obtained from 42 worm and cyst specimens from Sakha and, for comparison, from an additional 13 G10 cysts from Finland. For comparative analyses of the genetic diversity of G10 of European and Asian origin, all available cox1 sequences from GenBank were included, increasing the number of sequences to 99. The diversity found in northeast Asia was by far higher than in Europe, suggesting that the geographic origin of E. canadensis (at least of G10) might be northeast Asia.

Recently, there have been epidemics of human cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Kyrgyzstan. This study investigated 2 districts for the presence of Echinococcus granulosus s.l. and Echinococcus multilocularis eggs; species identity was confirmed by polymerase chain reaction in dog feces and the level of environmental contamination with parasite eggs in 2017–2018 was also investigated. In the Alay district 5 villages with a high reported annual incidence of AE of 162 cases per 100 000 and 5 villages in the Kochkor district which had a much lower incidence of 21 cases per 100 000 were investigated. However, the proportion of dog feces containing E. granulosus s.l. eggs was ~4.2 and ~3.5% in Alay and Kochkor respectively. For E. multilocularis, the corresponding proportions were 2.8 and 3.2%. Environmental contamination of Echinococcus spp. eggs was estimated using the McMaster technique for fecal egg counts, weight and density of canine feces. The level of environmental contamination with E. multilocularis eggs was similar at 4.4 and 5.0 eggs per m2 in Alay and Kochkor respectively. The corresponding values for E. granulosus s.l. were 8.3 and 7.5 eggs per m2. There was no association between village or district level incidence of human AE or CE and the proportion of dog feces containing eggs of Echinococcus spp. or the level of environmental contamination. Increased contamination of taeniid eggs occured in the autumn, after the return of farmers with dogs from summer mountain pastures.

The alveolar hydatid disease, also known as alveolar echinococcosis, of humans is certainly one of the most dangerous zoonoses worldwide. The disease is caused by Echinococcus multilocularis – the fox tapeworm. Red foxes (Vulpes vulpes) are currently counted as the most important carriers (reservoirs) of E. multilocularis in the Northern Hemisphere. The possible routes of infection of E. multilocularis to humans are complex and still require research. Until now, it has been unknown whether E. multilocularis eggs can be moved by wind at all. This analysis shows, based on calculations, that E. multilocularis eggs can be transported by wind. Using a mathematical model, flight distances depending on wind speed and take-off heights are calculated for dense and less dense (coniferous) forest areas. The results – differentiated for seasons and as overall average – are based on mean values of wind speeds which were measured over a ten-year period in an experimental forest stand in the Solling (Germany). Due to their rate of descent, wind-related spreading of E. multilocularis eggs is possible. The average flight distance covered by E. multilocularis eggs in forest areas, depending on their starting altitude and wind speed, is between approximately 1.3 m and approximately 17 m. From the mathematical point of view, the wind factor can definitely be seen as one of the multiple vectors associated with environmental contamination by E. multilocularis eggs. Consequently, the possible wind-borne spread of E. multilocularis eggs poses an infection risk to humans that should be considered and requires further research.

In a fast-changing and globalized world, parasites are moved across continents at an increasing pace. Co-invasion of parasites and their hosts is leading to the emergence of infectious diseases at a global scale, underlining the need for integration of biological invasions and disease ecology research. In this review, the ecological and evolutionary factors influencing the invasion process of parasites with complex life cycles were analysed, using the invasion of the European strain of Echinococcus multilocularis in North America as a model. The aim was to propose an ecological framework for investigating the invasion of parasites that are trophically transmitted through predator–prey interactions, showing how despite the complexity of the cycles and the interactions among multiple hosts, such parasites can overcome multiple barriers and become invasive. Identifying the key ecological processes affecting the success of parasite invasions is an important step for risk assessment and development of management strategies, particularly for parasites with the potential to infect people (i.e. zoonotic).

Susceptibility to Echinococcus multilocularis infection considerably varies among intermediate (mostly rodents) and dead-end host species (e.g. humans and pig), in particular regarding intestinal oncosphere invasion and subsequent hepatic metacestode development. Wistar rats are highly resistant to infection and subsequent diseases upon oral inoculation with E. multilocularis eggs, however, after immunosuppressive treatment with dexamethasone, rats become susceptible. To address the role of the cellular innate immunity, Wistar rats were individually or combined depleted of natural killer (NK) cells, macrophages (MΦ) and granulocytes (polymorphonuclear cells, PMN) prior to E. multilocularis egg inoculation. Although NK cell and MΦ depletion did not alter the resistance status of rats, the majority of PMN-depleted animals developed liver metacestodes within 10 weeks, indicating that PMN are key players in preventing oncosphere migration and/or development in Wistar rats. In vitro studies indicated that resistance is not caused by neutrophil reactive oxygen species or NETosis. Also, light microscopical examinations of the small intestine showed that oral inoculation of E. multilocularis eggs does not elicit a mucosal neutrophil response, suggesting that the interaction of oncospheres and neutrophils may occur after the former have entered the peripheral blood. We suggest to consider granulocytes as mediators of resistance in more resistant species, such as humans.

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is an important emerging parasite in the northern hemisphere. In epidemiological studies, the highest attention is being paid to foxes as the main reservoir hosts responsible for geographic expansion from multiple focal populations and the invasion of urban habitats, but little information is available on the parasite distribution in other carnivores. Hence, the study was designed to obtain updated information about the occurrence and genetic diversity of E. multilocularis in grey wolves and dogs in Slovakia. Faecal samples of wolves were collected from three locations under a certain level of environmental protection in the central and eastern parts of the country, and the presence of the parasite DNA was detected in 35.7% of 112 samples, with the highest rate (51.2%) recorded in the Poloniny National Park in north-eastern Slovakia. Among 110 faecal dog samples, E. multilocularis was detected in three faeces from segregated Roma settlements in the eastern part of the country, which accounted for an overall positivity of 2.7%. Sequence analysis of two mitochondrial genes, 12S rRNA and NADH dehydrogenase subunit 1, revealed four haplotypes in 13 isolates from wolves and dogs originating from four sites in eastern and central Slovakia, with all samples bearing a European-type pattern of E. multilocularis. The more than one-third positivity rate of E. multilocularis in wolf faecal samples dispersed over a large part of the country has corroborated the extensive circulation of the parasite in wildlife and confirmed the need to improve intervention control strategies.

The metacestode of Echinococcus multilocularis is the etiological agent of alveolar echinococcosis. The metacestode stage used for research is maintained in rodents by serial passages. In order to determine whether cryopreservation of E. multilocularis metacestodes would be suitable for long-term maintenance and replace serial passages, isolates of different geographic origin were cryopreserved in 1984–1986. The aim of the current study was to test the viability of cryopreserved isolates following long-term cryopreservation (up to 35 years) and to determine the phylogenetic clades these isolates belonged to. Cryopreserved isolates were tested for viability in vitro and in vivo in gerbils. In vitro results of 5 isolates indicated protoscolex survival in 13 of 17 experiments (76%) and metacestode survival in 5 of 12 (42%) in vivo experiments. In vivo results showed ‘abortive lesions’ in 13 of the 36 animals, 15 were negative and 8 harboured proliferating metacestode tissue containing protoscoleces. Genetic analysis confirmed the isolates belonged to European, Asian and North-American clades. In conclusion, the results of the current study indicate that metacestodes of E. multilocularis are able to survive long-term cryopreservation. Therefore, cryopreservation is a suitable method for long-term storage of E. multilocularis metacestode isolates and reduces the number of experimental animals.

Alveolar echinococcosis is a neglected parasitic zoonosis caused by Echinococcus multilocularis. The pharmacological treatment is based on albendazole (ABZ). However, the low water solubility of the drug produces a limited dissolution rate, with the consequent failure in the treatment of the disease. Solid dispersions are a successful pharmacotechnical strategy to improve the dissolution profile of poorly water-soluble drugs. The aim of this work was to determine the in vivo efficacy of ABZ solid dispersions using poloxamer 407 as a carrier (ABZ:P407 solid dispersions (SDs)) in the murine intraperitoneal infection model for secondary alveolar echinococcosis. In the chemoprophylactic efficacy study, the ABZ suspension, the ABZ:P407 SDs and the physical mixture of ABZ and poloxamer 407 showed a tendency to decrease the development of murine cysts, causing damage to the germinal layer. In the clinical efficacy study, the ABZ:P407 SDs produced a significant decrease in the weight of murine cysts. In addition, the SDs produced extensive damage to the germinal layer. The increase in the efficacy of ABZ could be due to the improvement of water solubility and wettability of the drug due to the surfactant nature of poloxamer 407. In conclusion, this study is the basis for further research. This pharmacotechnical strategy might in the future offer novel treatment alternatives for human alveolar echinococcosis.

Alveolar echinococcosis (AE) is a zoonotic parasitic diseases caused by a cestode parasite known as Echinococcus multilocularis. The parasite has a wildlife cycle with definitive hosts (foxes) and small mammals as intermediate hosts (rodents) while humans are the accidental hosts. Parasite infection pressure relation to time of the year and age dependent infection pressure for parasite abundance also depend on the urbanization. The aim of current work is forecasting the thresholds via the computational analysis of the disease spread which is a useful approach since it can help to design the experimental settings with better planning and efficiency. Network analysis when interlinked with the computational techniques provides better insight into the spatial and temporal heterogeneities. In the present study, a mathematical framework that describes the transmission dynamics and control measures of E. multilocularis in foxes is documented. We used treatment of foxes with baits for the prevention of the E. multilocularis infection. A novel approach of networking, called Petri net (PN), based on density dependent differential equations, is utilized during this research. The accurate description of the transmission of the parasite and the effect of drug on it is provided to the readers in this article. The transitions, which are difficult to analyse theoretically, are presented with the aid of the discrete approach of networking. A discrete mathematical framework can prove to be an accurate and robust tool to analyse and control the parasite dynamics.

Alveolar echinococcosis is a neglected parasitic zoonosis caused by the metacestode Echinococcus multilocularis, which grows as a malignant tumour-like infection in the liver of humans. Albendazole (ABZ) is the antiparasitic drug of choice for the treatment of the disease. However, its effectiveness is low, due to its poor absorption from the gastro-intestinal tract. It is also parasitostatic and in some cases produces side-effects. Therefore, an alternative to the treatment of this severe human disease is necessary. In this context, the repositioning of drugs combined with nanotechnology to improve the bioavailability of drugs emerges as a useful, fast and inexpensive tool for the treatment of neglected diseases. The in vitro and in vivo efficacy of dichlorophen (DCP), an antiparasitic agent for intestinal parasites, and silica nanoparticles modified with DCP (NP-DCP) was evaluated against E. multilocularis larval stage. Both formulations showed a time and dose-dependent in vitro effect against protoscoleces. The NP-DCP had a greater in vitro efficacy than the drug alone or ABZ. In vivo studies demonstrated that the NP-DCP (4 mg kg−1) had similar efficacy to ABZ (25 mg kg−1) and greater activity than the free DCP. Therefore, the repurposing of DCP combined with silica nanoparticles could be an alternative for the treatment of echinococcosis.

Recently, we introduced an epoxy group to mebendazole by a reaction with epichlorohydrin and obtained two isoforms, mebendazole C1 (M-C1) and mebendazole C2 (M-C2). The in vitro effects of mebendazole derivatives at different concentrations on Echinococcus multilocularis protoscoleces and metacestodes as well as cytotoxicity in rat hepatoma (RH) cells were examined. The results demonstrated that the solubility of the two derivatives was greatly improved compared to mebendazole. The mortality of protoscoleces in vitro reached to 70–80% after 7 days of exposure to mebendazole or M-C2, and M-C2 showed higher parasiticidal effects than mebendazole (P > 0.05). The parasiticidal effect of M-C1 was low, even at a concentration of 30 µm. The percentage of damaged metacestodes that were treated with mebendazole and M-C2 in vitro at different concentrations were similar, and M-C1 exhibited insignificant effects on metacestodes. Significant morphological changes on protoscoleces and metacestodes were observed after treatment with mebendazole and M-C2. In addition, the introduction of an epoxy group to mebendazole also reduced its cytotoxicity in RH cells. Our results demonstrate that the introduction of an epoxy group not only improved the solubility of mebendazole, but also increased its parasiticidal effects on E. multilocularis and reduced its cytotoxicity in RH cells.

This study investigated Echinococcus genotypes in patients with hydatidosis that reside in Inner Mongolia, Tibet or Qinghai Province by partially sequencing the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) genes. Cyst fluids were collected from 23 patients with hydatidosis and DNA was extracted. Portions of the cox1 and nad1 genes were amplified and subsequently sequenced. Sequencing analysis determined that one of the isolates contained Echinococcus multilocularis, and the other 22 isolates contained E. granulosus sensu lato. The isolates were then further classified based on genotype, and E. granulosus sensu stricto (s.s.) G1 (n = 20), E. granulosus s.s. G3 (n = 1) and E. canadensis G6/7 (n = 1) were identified. Additionally, the sequences were concatenated (pcox1 + pnad1) and 11 haplotypes were identified among the E. granulosus s.s. isolates (G1 and G3), with a shared common haplotype (H1) identified. Overall, these findings provide further understanding of the genetic patterns of Echinococcus in western and northern China.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most threatening zoonoses in Eurasia. Human AE is widespread in the Erzurum region of Turkey, but the situation of the disease in intermediate and definitive hosts is unknown. A Eurasian lynx (Lynx lynx) was killed in a traffic accident in the north of Erzurum, and was taken to our laboratory. Sedimentation and counting technique (SCT), DNA isolation and polymerase chain reaction (PCR) analysis were performed. The SCT results showed that the lynx was infected with E. multilocularis with a medium (745 worms) worm burden. The DNA of adult worms obtained from the lynx was analyzed with a species-specific PCR, and the worms were confirmed to be E. multilocularis by 12S rRNA gene sequence analysis. This is the first report of E. multilocularis from Eurasian lynx in Turkey.

MicroRNAs (miRNAs) are short noncoding RNAs, involved in the regulation of parasite diseases. However, a role of miRNAs in Echinococcus multilocularis infection remains largely unknown. In this study, we first found the expression levels of key genes involved in miRNA biogenesis and function, including Ago2, Xpo5, Tarbp2 and DgcR8, were obviously altered in the macrophage RAW264·7 cells exposed to E. multilocularis metacestodes. Compared with the control, 18 and 32 known miRNAs were found to be differentially expressed (P < 0·05 and fold change >2) in the macrophages exposed to E. multilocularis metacestodes for 6 and 12 h, respectively. Among these, several are known to be involved in regulating cytokine activities and immune responses. Quantitative real-time polymerase chain reaction results showed that the expression of nine selected miRNAs was consistent with the sequencing data at each treatment time points. Moreover, there were statistically significant correlations between the expression levels of miRNAs and their corresponding targeted genes. Our data give us some clues to pinpoint a role of miRNAs in the course of infection and immunity of E. multilocularis.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a potentially fatal zoonotic disease. Large parts of Turkey are considered as endemic for E. multilocularis. The aim of this study was to determine the occurrence of metacestode of E. multilocularis in wild rodents in Erzurum, an endemic region for human AE in Turkey. During the sampling period, a total of 498 rodents were trapped in twenty counties of Erzurum Province. Suspected lesions were observed on the livers of 48 rodents, and then partial fragment of mitochondrial 12S rRNA gene was PCR-amplified. Five liver samples exhibited E. multilocularis infection. The prevalence of E. multilocularis for Microtus spp. was 1·3%. All of the infected rodents had fertile metacestodes. Infected rodents were morphologically and molecularly analysed and were confirmed to be Microtus irani by the mitochondrial cytochrome b gene sequence analysis. This is the first report of the presence of E. multilocularis in rodent intermediate hosts in Turkey. Our findings of infected M. irani with protoscoleces show that this rodent can act as suitable intermediate host for E. multilocularis’ life cycle in Turkey. However, there was a complete lack of data on the infection of carnivores from the country. An extensive survey is recommended to determine the prevalence of E. multilocularis in definitive hosts in this endemic region.

Although local prevalence of Echinococcus multilocularis may be high, this zoonotic parasite has an overall low prevalence in foxes and rodents in Sweden. To better understand opportunities for E. multilocularis transmission in the Swedish environment, the aim of this study was to investigate other taeniid cestodes and to relate observed patterns to E. multilocularis. Cestode parasites were examined in fox feces and rodents caught in different habitats from four regions of Sweden. Arvicola amphibius and Microtus agrestis were parasitized with Versteria mustelae, Hydatigera taeniaeformis s. l., and E. multilocularis, whereas Myodes glareolus and Apodemus spp. were parasitized with V. mustelae, Taenia polyacantha, H. taeniaeformis s.l., and Mesocestoides spp. Rodents caught in field habitat (Ar. amphibius, Mi. agrestis) were more likely (OR 10, 95% CI 5–19) to be parasitized than rodents caught in forest habitat (My. glareolus, Apodemus spp.). The parasite preference for each rodent species was present regardless of the type of background contamination from fox feces. These results further support the importance of both ecological barriers and individual species susceptibility in parasite transmission, and indicate that future monitoring for E. multilocularis in the Swedish environment should focus in field habitats where Mi. agrestis and Ar. amphibius are abundant.

Echinococcus multilocularis is a most pathogenic zoonotic tapeworm that causes devastating echinococcosis in both humans and animals. Circulating microRNAs (miRNAs) are stably existed in the serum/plasma of mammalian hosts during helminthic infection. In this study, we compared the host-circulating miRNA expression in the sera from the E. multilocularis-infected and uninfected mice. A total of 58 host-origin serum miRNAs were differentially expressed (2 ⩾ fold change, P < 0·05), of which 21 were upregulated and 37 were significantly downregulated. Consistent with the sequencing data, quantitative polymerase chain reaction (PCR) results showed that the expression levels of four miRNAs were elevated gradually and one decreased gradually at the E. multilocularis infection time points. Moreover, seven of E. multilocularis specific miRNAs were identified in the sera. Real-time PCR analyses further demonstrated that only two parasite-derived miRNAs (emu-miR-10 and emu-miR-227) were specifically amplified in all the sera from mice infected with E. multilocularis. These findings will be helpful to understand the roles of miRNAs in host–parasite interaction and to potentiate serum miRNAs as diagnostic targets for echinococcosis.