First description of the emergence of Echinococcus ortleppi (G5 genotype) in sheep and goats in Iran.

Cystic echinococcosis is one of the most important cyclozoonotic helminthic diseases caused by various genotypes of Echinococcus granulosus SENSU lato complex in the Middle East. Echinococcus ortleppi G5 genotype has been previously reported to infect camels in Iran. However, no molecular data are available on the circulation of the cattle genotype from other animal intermediate hosts. Overall, 30 hydatid cyst samples were collected from 2015 to 2016 from sheep (n; 15) and goat (n; 15) isolates in Mazandaran province, northern Iran. The DNA of larval stages was extracted, amplified and sequenced by targeting of mitochondrial 12S rRNA marker. Based on sequencing and phylogenetic analyses, 28 isolates were identified as E. granulosus G1. However, two fertile hydatid cyst samples isolated from sheep and goat liver were confirmed as E. ortleppi with 99.8%-100% identity to species isolated from a buffalo in India. The first emergence of fertile cattle cysts from sheep and goats may have implications in the zoonotic importance of E. ortleppi in human infection and de-worming regime of infected dogs due to the shorter maturation time of G5 genotype compared with G1 genotype. Since the distribution of E. ortleppi in Iran is not fully known, further studies are urgently needed to appraise the evolutionary and epidemiology scenarios. These studies can ascertain the transmission dynamics of E. ortleppi inferred by full-length concatenated mitogenomes on the domestic and wildlife hosts, as well as humans in wider areas of Iran and the neighboring countries.

Effect of Taxus baccata L. Extract on Hydatid Cyst Protoscolices In vitro.

Hydatidosis is the most important global parasitic infectious disease both in humans and animals, which can lodge at different organs of the host, such as liver, lung (even heart), and brain which may lead to death. Surgery is the main method for the treatment of hydatidosis. In surgical therapy of hydatidosis, the use of sporicidal agents is very important since these agents inactivate live protoscolices and prevent recurrence of infection. Presently, numerous scolicidal chemical agents have been administrated to inactivate the hydatid cyst contents. Recently, there has been a high tendency among researchers to evaluate and present herbal plants as alternative option due to inexpensiveness, availability, low side effects, and toxicity. This study aimed to evaluate the scolicidal effect of hydro alcoholic Taxus baccata L. extract in vitro for the first time. The scolicidal activities of the extract were tested in concentrations of 50, 100, and 150 mg/ml following 10, 30, and 60 min of incubation, and the experiments were performed in triplicate. Viability of protoscolices was confirmed by 0.1% eosin vital staining. The data were analyzed in SAS software (version 9.4). The results showed that the hydroalcoholic extract of Taxus baccata L. at the concentration of 150 mg/ml led to killing 66.6% of protoscolices at 60 min. according to the results of this investigation, it is recommended to use this plant as a scolicidal plant. The findings of the present study showed that Taxus baccata L. had potent scolicidal effects. However, further studies are required to evaluate the efficacy of Taxus baccata L. in vivo.

Peritoneal Hydatid Cysts in Children: A Case Series of Rare Echinococcosis Localization.

Peritoneal hydatid cysts are rare in children even in endemic areas. The primary or secondary origin of this site remains controversial, especially in children. Secondary peritoneal hydatid cysts are mainly the result of spontaneous or traumatic rupture of concomitant liver cysts or the leakage of cystic content during surgery. The purpose of our study is to present the largest case series of peritoneal hydatidosis in children. In addition, we aimed to assess the clinical and paraclinical findings as well as the management of echinococcosis at this location in children. The present case series is a study of ten children with peritoneal hydatid cysts, who underwent surgical intervention between 2013 and 2018 in the Pediatric Surgery Department, University of Monastir (Monastir, Tunisia). The mean age of the children was six years. All children presented abdominal pain, and underwent ultrasonography and contrast-enhanced computed tomography of the abdomen. Two patients had been operated on for lung hydatid cysts six months prior to the study. In two cases, radiologic investigations revealed the presence of an uncomplicated hepatic hydatid cyst located in segments II and IV. All patients underwent surgery, of which four underwent laparoscopy. Post-surgery, all patients received albendazole for three months, and the follow-up period was uneventful. Currently, all patients are in good health. Peritoneal hydatid disease is frequently secondary to the rupture of a primary hepatic cyst. Diagnosis is performed by abdominal ultrasound, computed tomography, and a positive serology result. Open or laparoscopic excision can be combined with medical treatment.

Dihydroartemisinin induces ER stress-dependent apoptosis of Echinococcus protoscoleces in vitro.

In this study, we investigated the effect of dihydroartemisinin on Echinococcus protoscoleces and explored the role of endoplasmic reticulum stress in this process. Echinococcus protoscoleces were collected and cultured in RPMI 1640 medium. Changes in the expressions of glucose-regulated protein 78 (GRP-78), caspase-12, and C/EBP homologous protein (CHOP) were assessed through confocal immunofluorescence and western blot analysis. Cell viability and morphological changes were observed under a light microscope. The ultrastructure of protoscoleces was observed by scanning electron microscopy and transmission electron microscopy. Caspase-3 activity was detected using an enzyme assay kit. After dihydroartemisinin treatment, the protoscoleces showed loss of viability, and morphological changes including soma contraction, blebs formation, hooks loss, microtrichia destruction, and development of lipid droplets was observed. The levels of caspase-12 and CHOP were increased within 2 days of dihydroartemisinin treatment. However, the levels of GRP-78, caspase-12, and CHOP were decreased in 4 days. Furthermore, caspase-3 activity was increased after treatment with different concentrations of dihydroartemisinin. Dihydroartemisinin can induce apoptosis in protoscoleces via the ER stress-caspase-3 apoptotic pathway in vitro. These results indicate that dihydroartemisinin is a potentially valuable therapeutic agent against echinococcosis.

In vivo treatment with IL-17A attenuates hydatid cyst growth and liver fibrogenesis in an experimental model of echinococcosis.

We aimed to assess the effect of exogenous Interleukin (IL)-17A in experimental model of echinococcosis. Swiss mice were inoculated intra-peritoneally with viable protoscoleces (PSCs). Then, IL-17A was administered at 100, 125 or 150 pg/mL two weeks after cystic echinococcosis (CE) induction. Cyst development and hepatic damage were macroscopically and histologically analyzed. We observed that in vivo IL-17A treatment at 100, 125, and 150 pg/mL, reduced metacestode growth by 72.3%, 93.8%, and 96.9%, respectively. Interestingly an amelioration of liver architecture was noted at 125 pg/mL without toxic effect. In this context, we showed less fibrosis reaction and reduced expression of iNOS, TNF-α, NF-κb and CD68 in hepatic parenchyma of treated mice by 125 pg/mL of IL-17A. Collectively, our results indicate an antihydatic effect and immunoprotective properties of IL-17A and suggest its potential therapeutic value against Echinococcus granulosus infection.

Genome-wide identification of microRNA targets in the neglected disease pathogens of the genus Echinococcus.

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in biological processes such as development. MiRNAs silence target mRNAs by binding to complementary sequences in the 3'untranslated regions (3'UTRs). The parasitic helminths of the genus Echinococcus are the causative agents of echinococcosis, a zoonotic neglected disease. In previous work, we performed a comprehensive identification and characterization of Echinococcus miRNAs. However, current knowledge about their targets is limited. Since target prediction algorithms rely on complementarity between 3'UTRs and miRNA sequences, a major limitation is the lack of accurate sequence information of 3'UTR for most species including parasitic helminths. We performed RNA-seq and developed a pipeline that integrates the transcriptomic data with available genomic data of this parasite in order to identify 3'UTRs of Echinococcus canadensis. The high confidence set of 3'UTRs obtained allowed the prediction of miRNA targets in Echinococcus through a bioinformatic approach. We performed for the first time a comparative analysis of miRNA targets in Echinococcus and Taenia. We found that many evolutionarily conserved target sites in Echinococcus and Taenia may be functional and under selective pressure. Signaling pathways such as MAPK and Wnt were among the most represented pathways indicating miRNA roles in parasite growth and development. Genome-wide identification and characterization of miRNA target genes in Echinococcus provide valuable information to guide experimental studies in order to understand miRNA functions in the parasites biology. miRNAs involved in essential functions, especially those being absent in the host or showing sequence divergence with respect to host orthologs, might be considered as novel therapeutic targets for echinococcosis control.

Ecology and Life Cycle Patterns of Echinococcus Species.

The genus Echinococcus is composed of eight generally recognized species and one genotypic cluster (Echinococcus canadensis cluster) that may in future be resolved into one to three species. For each species, we review existing information on transmission routes and life cycles in different geographical contexts and - where available - include basic biological information of parasites and hosts (e.g., susceptibility of host species). While some Echinococcus spp. are transmitted in life cycles that involve predominantly domestic animals (e.g., dog - livestock cycles), others are wildlife parasites that do or do not interact with domestic transmission. In many cases, life cycle patterns of the same parasite species differ according to geography. Simple life cycles contrast with transmission patterns that are highly complex, involving multihost systems that may include both domestic and wild mammals. Wildlife transmission may be primary or secondary, i.e., resulting from spillovers from domestic animals. For most of the species and regions, existing information does not yet permit a conclusive description of transmission systems. Such data, however, would be highly relevant, e.g., for anticipation of geographical changes of the presence and frequency of these parasites in a warming world, or for initiating evidence-based control strategies.

Biology and Systematics of Echinococcus.

The biology of Echinococcus, the causative agent of echinococcosis (hydatid disease) is reviewed with emphasis on the developmental biology of the adult and metacestode stages of the parasite. Major advances include determining the origin, structure and functional activities of the laminated layer and its relationship with the germinal layer; and the isolation, in vitro establishment and characterization of the multipotential germinal cells. Future challenges are to identify the mechanisms that provide Echinococcus with its unique developmental plasticity and the nature of activities at the parasite-host interface, particularly in the definitive host. The revised taxonomy of Echinococcus is presented and the solid nomenclature it provides will be essential in understanding the epidemiology of echinococcosis.

First study about the development of adult Echinococcus canadensis G6 genotype of goat origin in experimentally infected dogs.

Echinococcus granulosus sensu lato (E. granulosus sl) must be considered as a species complex, comprising Echinococcus granulosus sensu stricto (E. granulosus ss, genotypes G1-G3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and Echinococcus canadensis (G6-G10) although the species status of E. canadensis is still controversial. These genotypes closely match the intermediate hosts associated strains described in earlier times among which E. canadensis G6 corresponds to the camel strain. As there are no studies concerning the development of adult stages of the G6 genotype from non-camel origin, the aims of the present study were: to characterize for the first time the development of E. canadensis G6 in dogs experimentally infected with protoscoleces derived from goats, to describe the resultant adult morphology, to evaluate the growth of their rostellar hooks from larval to adult stages and to determine the prepatent period of the strobilar stage of E. canadensis G6 derived from goats. The development of the strobilar stage of E. canadensis G6 genotype of goat origin was examined by studying the growth (variation of the total worm length) and segmentation in experimentally infected dogs at 14, 25, 35 and 56days post infection. A morphological characterization of 35-day-old worms as well as of larval and adult rostellar hooks was also carried out by conventional optical microscopic observations and/or by scanning electron microscopy. The prepatent period of the strobilar stage was assessed by microscopic examination of faeces from 2 infected dogs. Our results were compared with published data from the camel and other strains. The roles of the host, genotype and species in morphological and developmental features as well as the taxonomic position of E. canadensis G6 were discussed. The prepatent period of E. canadensis G6 genotype of goat origin was determined as at least, 41days. The obtained results contribute to increase the knowledge about the biology and genetics of E. granulosus sl complex and are also of practical usefulness for the design of disease control strategies.

Correlation of serum sHLA-G levels with cyst stage in patients with cystic echinococcosis: is it an immune evasion strategy?

Patients with cystic echinococcosis (CE) can harbour cysts for years or even decades, apparently without effect of the immune system on the metacestode. Although several immune evasion mechanisms by echinococcal cysts have been described, it is unclear whether the human leucocyte antigen (HLA) system plays a role in the susceptibility or resistance to CE in humans. HLA-G molecules are known to exert a suppressive action on dendritic cells maturation and on natural killer (NK) cells functions, therefore hampering T-cell responses and NK cytolysis. HLA-G plays an important role in immune tolerance, is involved in foetus and in allotransplant tolerance, and may be involved in tumoral and viral immune evasion. In this study, we assessed the presence and levels of soluble HLA-G (sHLA-G) in patients with CE using a commercial ELISA kit to determine whether host's HLA-G may have a role in the course of human CE.

Fine-needle aspiration diagnosis of primary hydatid disease of the thyroid; first reported case in the USA.

Echinococcosis or hydatid disease (HD) is a parasitic disease caused by species of the Echinococcus genus. Since the incidence of HD in the USA is very low and the primary HD of the thyroid is extremely rare even in endemic regions, the occurrence of primary thyroid HD is exceptional in the USA. Thyroid HD is rarely diagnosed by fine-needle aspiration (FNA). Our literature review revealed less than ten cases of primary HD of thyroid diagnosed by FNA worldwide. Hereby, we report the first case of a primary thyroid HD diagnosed by fine-needle aspiration in the USA.

Efficacy of Myrtus communis L. to Inactivate the Hydatid Cyst Protoscoleces.

PURPOSE: The present study aims to investigate the scolicidal effects of Myrtus communis L. essential oil against protoscoleces of hydatid cysts and also its toxicity in mice model. MATERIALS AND METHODS: Protoscoleces were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the essential oil (12.5-100 µl/ml) were used for 5-30 min. Viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining). Moreover, 48 male NMRI mice were used to determine the acute and sub-acute toxicity of M. communis essential oil. One-way ANOVA with Tukey's post-hoc test was used to assess differences between experimental groups. RESULTS: Findings of the present study demonstrated that the M. communis essential oil at the concentration of 100 µl/ml after 5 min of exposure killed 100% protoscoleces. Similarly, the mean mortality rate of protoscoleces after 10 min of exposure to concentration of 50 µl/ml was 100%. However, lower concentrations (12.5 and 25 µl/ml) of M. communis essential oil provoked a delayed protoscolicidal effects. The LD50 values of intraperitoneal injection of the M. communis essential oil was 2.23 mL/kg body wt. No significant difference (p > .05) was observed in the clinical chemistry and hematological parameters following oral administrations of M. communis essential oil at the doses 0.05, 0.1, 0.2, and 0.4 mL/kg for 14 days. CONCLUSION: The results showed potent scolicidal activity of M. communis with no significant toxicity, which might be used as a natural scolicidal agent in hydatid cyst surgery.

Report: Evaluation of the scolicidal effects of Nectaroscordum tripedale extract and its acute toxicity in mice model.

Current scolicidal agents, which have been used for inactivation of protoscoleces during hydatid cyst surgery are associated with adverse side effects. This study aims to evaluate the in vitro scolicidal effects of Nectaroscordum tripedale L. leave extract against protoscoleces of hydatid cysts and its acute toxicity in mice model. Various concentrations of the extract (12.5-100 mg/mL) were used for 5 to 30 min. Viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining). In addition, the acute toxicity of N. tripedale extract was determined for 2 days in mice model. The results showed that the N. tripedale extract at the concentration of 100 mg/mL after 5 min of exposure killed 100% protoscoleces. Similarly, the mean of mortality rate of protoscoleces after 10 min of exposure to concentration of 50 mg/mL was 100%. The LD50 values of intraperitoneal injection of the N. tripedale extract was 3.36 g/kg body wt. and the maximum nonfatal doses were 2.98 g/kg body wt. The results showed the potential of N. tripedale extract as a natural source for the production of new scolicidal agent for use in hydatid cyst surgery.

Basic and applied problems in developmental biology and immunobiology of cestode infections: Hymenolepis, Taenia and Echinococcus.

Differentiation and development of parasites, including longevity in host animals, are thought to be governed by host-parasite interactions. In this review, several topics on the developmental biology of cestode infections are discussed from immunobiological perspective with a focus on Hymenolepis, Taenia and Echinococcus infections. The basic premise of this review is that 'differentiation and development of cestodes' are somehow affected by host immune responses with an evolutionary history.

On the importance of targeting parasite stem cells in anti-echinococcosis drug development.

The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemotherapy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating proliferation of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite's stem cell population.

Echinococcus metacestode: in search of viability markers.

Epidemiological studies have demonstrated that most humans infected with Echinococcus spp. exhibit resistance to disease. When infection leads to disease, the parasite is partially controlled by host immunity: in case of immunocompetence, the normal alveolar echinococcosis (AE) or cystic echinococcosis (CE) situation, the metacestode grows slowly, and first clinical signs appear years after infection; in case of impaired immunity (AIDS; other immunodeficiencies), uncontrolled proliferation of the metacestode leads to rapidly progressing disease. Assessing Echinococcus multilocularis viability in vivo following therapeutic interventions in AE patients may be of tremendous benefit when compared with the invasive procedures used to perform biopsies. Current options are F18-fluorodeoxyglucose-positron emission tomography (FDG-PET), which visualizes periparasitic inflammation due to the metabolic activity of the metacestode, and measurement of antibodies against recEm18, a viability-associated protein, that rapidly regresses upon metacestode inactivation. For Echinococcus granulosus, similar prognosis-associated follow-up parameters are still lacking but a few candidates may be listed. Other possible markers include functional and diffusion-weighted Magnetic Resonance Imaging (MRI), and measurement of products from the parasite (circulating antigens or DNA), and from the host (inflammation markers, cytokines, or chemokines). Even though some of them have been promising in pilot studies, none has been properly validated in an appropriate number of patients until now to be recommended for further use in clinical settings. There is therefore still a need to develop reliable tools for improved viability assessment to provide the sufficient information needed to reliably withdraw anti-parasite benzimidazole chemotherapy, and a basis for the development of new alternative therapeutic tools.

Echinococcus as a model system: biology and epidemiology.

The introduction of Echinococcus to Australia over 200 years ago and its establishment in sheep rearing areas of the country inflicted a serious medical and economic burden on the country. This resulted in an investment in both basic and applied research aimed at learning more about the biology and life cycle of Echinococcus. This research served to illustrate the uniqueness of the parasite in terms of developmental biology and ecology, and the value of Echinococcus as a model system in a broad range of research, from fundamental biology to theoretical control systems. These studies formed the foundation for an international, diverse and ongoing research effort on the hydatid organisms encompassing stem cell biology, gene regulation, strain variation, wildlife diseases and models of transmission dynamics. We describe the development, nature and diversity of this research, and how it was initiated in Australia but subsequently has stimulated much international and collaborative research on Echinococcus.