Morphology of Echinococcus granulosus protoscolex / Morfología de los Protoescolex de echinococcus granulosus

SUMMARY: Echinococcus granulosus (E. granulosus), is a tapeworm that spreads between intermediate and definitive hosts through the ingestion of fecal matter contaminated with the parasite's eggs. The life cycle consists of differentiation from eggs to oncospheres to embryos, which eventually form cysts in organs like the liver, lungs and others. Within these cysts are protoscolices, an intermediate stage of the parasite which develop into adult tapeworms once they infect their definitive hosts. When these hydatid cysts form in humans, it is known as Cystic Echinococcosis (CE). This disease is treated through surgical excision of the cysts and or chemotherapy with benzimidazole compounds. Understanding the morphology of the intermediate developmental stage of E. granulosus, protoscolex stage, can allow researchers to identify defining structural changes and protein functions that could be used to develop treatment modalities for CE. Unique characteristics in the tegumental surface during the protoescolex stage and proteins associated with cyst fertility have all been identified in previous research studies and bring researchers closer to understanding the underlying mechanisms of E. granulosus development, and consequently, means to disrupt it to achieve better control of the disease.

El Echinococcus granulosus (E. granulosus), es un cestodo que se propaga entre huéspedes intermedios y definitivos a través de la ingestión de materia fecal contaminada con los huevos del parásito. El ciclo de vida consiste en la diferenciación de huevos a oncosferas y embriones, que finalmente forman quistes en órganos como el hígado, los pulmones y otros. Dentro de estos quistes hay protoescólices, una etapa intermedia del parásito que se convierte en su forma adulta (tenia), una vez que infectan a sus huéspedes definitivos. Cuando estos quistes hidatídicos se desarrollan en seres humanos, se les conoce como equinococosis quística (EC). Esta enfermedad se trata mediante la extirpación quirúrgica de los quistes o la quimioterapia con compuestos benzimidazólicos. La comprensión de la morfología de la etapa de desarrollo intermedia del E. granulosus y la etapa de protosclex, puede permitir a los investigadores identificar cambios estructurales definidos y funciones de proteínas que podrían usarse para desarrollar modalidades de tratamiento para la CE. Las características únicas en la superficie tegumentaria durante la etapa de protoescolex y las proteínas asociadas con la fertilidad del quiste se han identificado en estudios de investigación anteriores y acercan a los investigadores a la comprensión de los mecanismos subyacentes del desarrollo del E. granulosus y, en consecuencia, los medios para interrumpirlo para lograr un mejor control de la enfermedad.

Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces.

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×107 PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3+ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.

Genome-wide transcriptome analysis of the early developmental stages of Echinococcus granulosus protoscoleces reveals extensive alternative splicing events in the spliceosome pathway.

BACKGROUND: The complex life cycle of Echinococcus granulosus involves numerous environmental conditions within different intermediate and definitive hosts. This requires adaptation at different levels of transcript regulation. Alternative splicing (AS) and the related cellular functions as one of the major fields of post-genomics has been poorly studied in tapeworms. In the present study, we investigated AS events and their potential biological effects in E. granulosus. METHODS: Whole transcriptome sequencing data of four groups of protoscoleces were prepared for RNA-seq library construction. Fresh protoscoleces were either used as non-induced controls (NT group) or incubated for 15 min with pepsin (PEP group) and cultivated in a biphasic medium for 12 and 24 h (12 and 24 h groups). The frequency and different types of AS events were identified using rMATS software. Functional annotations and gene ontology of differential AS (DAS) genes were performed using Blast2GO software. AS events were experimentally validated by PCR on the protoscolex cDNAs using specific primers for each gene. RESULTS: At least one AS event was found in 38.1% of the genes (3904 out of 10,245) in the protoscoleces during early strobilar development. The genes were associated primarily with cellular and metabolic processes and binding and catalytic activity. KEGG pathway analysis of DAS events revealed a number of genes belonging to different components of the spliceosome complex. These genes tended to belong to common SR proteins, U1-related factors, U2-related factors, complex A-specific factors and other splicing-related proteins. CONCLUSIONS: The high number of AS events in the transcriptome regulatory mechanisms indicates the essential rapid molecular processes required by the parasite for adaptation in different environments.

Protoscolicidal activity of Atriplex halimus leaves extract against Echinococcus granulosus protoscoleces.

Cystic echinococcosis, an endemic zoonosis in Algeria, is caused by the development of the helminth Echinococcus granulosus. Surgery remains the main treatment despite inducing relapse and several adverse reactions. In this context, natural scolicidal agents seem to be promising tools to overcome these reactions. In our study, we evaluated the phytochemical contents, antioxidant activity and scolicidal effect of Atriplex halimus. In this context, the aqueous extract from AH leaves (AHE) was subjected to preliminary phytochemical screening by HPLC. The in vitro antioxidant activity was determined by DPPH test. The cytotoxicity of AHE was evaluated in murine peritoneal macrophages and cell viability was examined by MTT assay. Moreover, different concentrations of AHE (20, 40, 50, 60 and 100 mg/ml) were tested on E. granulosus protoscoleces (PSC) cultures, during different times of incubation (15, 30, 60, 90, 120 and 180 min). The viability was evaluated by eosin exclusion test. The morphological and ultrastructural damages were evaluated by SEM. Our results indicate that total phenolic and flavonoids contents were 37.93 µg of Gallic acid equivalent per mg of extract (GAE/mg E) and 18.86 µg of Quercetin equivalent per mg (QE/mg E) respectively. Furthermore, AHE has an antioxidant activity with an IC50 of 0.95 mg/ml. Interestingly, the extracts did not exhibit any cytotoxic effect against murine peritoneal macrophages. Moreover, our study indicated a significant scolicidal activity time- and dose-dependent. At 60 and 100 mg/ml; and after 120 min of incubation; the mortality rate was 99.36 and 100%, respectively. The parasite's tegument is one of the plant's targets as demonstrated by SEM. Our findings show the benefits of Atriplex halimus extract as a new promising scolicidal tool in hydatid cyst treatment.

Haplotype comparisons of Echinococcus granulosus sensu lato via mitochondrial gene sequences (co1, cytb, nadh1) among Pakistan and its neighbouring countries.

Echinococcus granulosus sensu lato (s.l.) is a zoonotic parasite that causes cystic echinococcosis (CE) in humans. However, E. granulosus sensu stricto (s.s.) is considered the predominant species in CE infections worldwide. According to the population genetic diversity and structure of E. granulosus s.l., gene flow can explain the parasite drift among the neighbouring countries of Pakistan. The mitochondrial (mt) co1 (n = 47), nadh1 (n = 37) and cytb (n = 35) nucleotide sequences of E. granulosus s.l. isolates from Pakistan, Iran, China and India were retrieved from the National Centre for Biotechnology Information database to determine the genealogical relationships. The sequences were grouped as the mt-co1 (genotypes G1 and G3, G6-G7), mt-cytb (genotypes G1 and G3), and mt-nadh1(genotypes G1 and G3). The data were analysed using bioinformatic tools. A total of 19 polymorphic sites for the mt-co1 sequence (374 bp) were observed of which 31.6% (6/19) were parsimony-informative sites. Unique singleton haplotypes within the E. granulosus s.s. haplotype network based on the mt-co1 gene were highly prevalent (68.4%; 13/19) in Pakistani isolates followed by Chinese, Indian and Iranian isolates; four polymorphic sites were detected in the E. canadensis (G6/G7). In E. canadensis mt-co1 haplotype network, 75% (3/4) unique singleton haplotypes were from the Iranian isolates. Twelve polymorphic sites were found using the mt-cytb sequence (547 bp); 25% (3/12) were parsimony-informative and there were 66.7% (8/12) unique singleton haplotypes within the mt-cytb haplotype network in E. granulosus s.s. with the most reported from Pakistan followed by Iran and China. 20 polymorphic sites were detected in E. granulosus s.s. mt-nadh1 sequences (743 bp); 20% (4/20) were parsimony-informative. There were 66.7% (8/12) main single haplotypes within the mt-nadh1 haplotype network, with the most reported from Pakistan followed by that from India, Iran and China. The sequence analyses show low nucleotide diversity and high haplotype diversity in general.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Copper nanoparticles: Biosynthesis, characterization, and protoscolicidal effects alone and combined with albendazole against hydatid cyst protoscoleces.

BACKGROUND: Surgery remains the preferred treatment option for hydatid cyst (cystic echinococcosis); however, recent studies have demonstrated that the current protoscolicidal agents used during surgery are associated with some adverse side effects such as biliary fibrosis, hepatic necrosis, and cirrhosis. The present study aims to evaluate the in vitro and ex vivo anti-parasitic effects of copper nanoparticles (CuNPs) alone and combined with albendazole on hydatid cyst protoscoleces. METHODS: CuNPs was green synthesized using C. spinosa extract. Various concentrations of CuNPs (250, 500, and 750 mg/mL) alone and combined with albendazole (ALZ, 200 mg/mL) were exposed to protoscoleces collected from the liver fertile hydatid cysts of infected sheep for 5-60 min in vitro and ex vivo. Next, the eosin exclusion test was applied to determine the viability of protoscoleces. Caspase-3 like activity of CuNPs-treated protoscoleces was then evaluated using the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: Scanning electron microscopy (SEM) results showed that the particle size of CuNPs was 17 and 41 nm with the maximum peak at the wavelength of 414 nm. The maximum protoscolicidal activity of CuNPs was observed at the concentration of 750 mg/mL in vitro, so that 73.3 % of protoscoleces were killed after 60 min of exposure. Meanwhile, the mortality of protoscoleces was 100 % after 10 min of exposure to 750 mg/mL of CuNPs along with ALZ (200 mg/mL). Nevertheless, the findings proved that CuNPs even in combination with ALZ required a longer time to kill protoscoleces ex vivo. After 48 h of treating protoscoleces, CuNPs in a dose-dependent manner and at doses of 250, 500, and 750 mg/mL induced the caspase enzyme activation by 20.5 %, 32.3 %, and 36.1 %, respectively. CONCLUSION: The findings of the present investigation showed potent protoscolicidal effects of CuNPs, especially combined with albendazole, which entirely eliminated the parasite after 10-20 min of exposure. The results also showed that although the possible protoscolicidal mechanisms of CuNPs are not clearly understood, the inducing apoptosis through caspases is one of the main protoscolicidal mechanisms of CuNPs. However, supplementary studies, especially in animal models and clinical settings, are needed to approve these results.

First report of cystic echinococcosis caused by Echinococcus granulosus sensu stricto/G1 in Felis catus from the Patagonian region of Argentina.

Two domestic cats from the Patagonia rural area in Argentina were found to be naturally infected with Echinococcus granulosus sensu stricto/G1 genotype; so far, the only species/genotype of E. granulosus sensu lato complex described to infect domestic cats. The felines developed abdominal disseminated larval disease; the diagnosis was performed by ultrasound, exploratory laparotomy, and molecular techniques. These results indicate that cystic echinococcosis must be considered for differential diagnosis of felines with abdominal distension and/or observation of vesicles through ultrasound, from endemic areas. Even though cats and dogs are carnivores, differences in digestive physiology and immunological characteristics between them could allow the development of larval or adult worm parasites. Domestic cats with cystic echinococcosis show to be environmentally infected with E. granulosus s. s./G1 eggs.

In Vitro and In Vivo Efficacy of DNA Damage Repair Inhibitor Veliparib in Combination with Artesunate against Echinococcus granulosus.

Cystic echinococcosis (CE), caused by the cestode Echinococcus granulosus, is a worldwide chronic zoonosis. Albendazole (ABZ) and mebendazole are effective against CE, but a high dosage in a long-term period is usually required. In this study, we evaluate the effects of DNA damage repair inhibitor (i.e., Veliparib) in combination with artesunate (AS) on hydatid cysts. For the in vitro assay, protoscoleces of E. granulosus (E.g PSCs) were incubated with low AS (AS-L, 65 µM), moderate AS (AS-M, 130 µM), and high AS (AS-H, 325 µM), AS-L/M/H+Veliparib (10 µM), and ABZ (25 µM), respectively. The AS-H+Veliparib group showed the maximal protoscolicidal effects. Ultrastructural change revealed that germinal layer (GL) cells were reduced, and lipid droplets appeared. AS could induce DNA injuries in PSCs. The 8-OHdG was expressed in the PSCs and GL of the cysts in mice, especially in the presence of Veliparib. The most severe DNA damages were observed in the AS-H+Veliparib group. Meanwhile, the expression of ribosomal protein S9 (RPS9) gene in the AS-H+Veliparib group was significantly lower than that in the AS-H group. The in vivo chemotherapeutic effects of AS-L (50 mg/kg), AS-H (200 mg/kg), and AS-H+Veliparib (25 mg/kg) were assessed in experimentally infected mice. Upon 6 weeks of oral administration, ultrasonography was used to monitor the volume change of vesicles. Maximum potentiation was seen on day 15 with values (versus AS) of 34 (P < 0.05) for AS-H + Veliparib. It led to the reduction of cyst weight (55.40%) compared with the model group (P < 0.01), which was better than AS alone (52.84%) and ABZ-treated mice (55.35%). Analysis of cysts collected from AS-H+Veliparib-treated mice by transmission electron microscopy revealed a drug-induced structural destruction. The structural integrity of the germinal layer was lost, and the majority of the microtriches disappeared. In conclusion, our study demonstrates that AS or AS in combination with Veliparib is effective for treating CE, especially the combination group. On this basis, AS represented promising drug candidates in anti-CE chemotherapy.

In vitro efficacy study of Cinnamomum zeylanicum essential oil and cinnamaldehyde against the larval stage of Echinococcus granulosus.

Hydatidosis or cystic echinococcosis is a disease caused by the larval stage of Echinococcus granulosus sensu lato. Chemotherapy can be used alone or in combination with surgery or percutaneous treatment. Benzimidazoles are the only agents used and approved for treatment, but their efficacy is extremely variable. Therefore, it is necessary to find new drugs to improve the treatment of this disease. In the last decades, the biological properties of essential oils and their components began to be investigated as alternatives in the treatment of different ailments. The aim of the present work was to evaluate the in vitro efficacy of the essential oil of Cinnamomum zeylanicum (cinnamon) and cinnamaldehyde against protoscoleces and metacestodes of E. granulosus. The essential oil and cinnamaldehyde, its major component, showed a dose and time dependent effect against protoscoleces. However, cinnamaldehyde showed a greater protoscolicidal effect than the essential oil. The maximum protoscolicidal effect was found with 50 µg/mL of cinnamaldehyde. Viability decreased by 1.7 ± 0.8% after 4 days of incubation and reached 0% at 8 days. Interestingly, there were no significant differences between the activity of cinnamaldehyde at the concentrations of 25 and 10 µg/mL and the efficacy observed with the essential oil at 200 and 50 µg/mL, respectively. Cinnamaldehyde also had a strong in vitro effect against murine cysts, while only the higher concentration of the essential oil caused ultrastructural alterations. Working with components instead of with essential oils has some advantages, particularly in relation to the reproducibility of the formulations and their effectiveness. For this reason, the results obtained in this work are promising in the search for pharmaceutical alternatives for the treatment of cystic echinococcosis.

Genetic Affinity of Echinococcus granulosus protoscolex in Human and Sheep in East Azerbaijan, Iran.

Echinococcosis caused by the larval form of Echinococcus granulosus (E. granulosus) is known as an important zoonotic disease in various parts of the world, including Iran. The genetic diversity of this parasite is very high, particularly in areas where the disease is endemic. It has been suggested in the literature from different parts of the world that diverse factors, such as parasite life cycle, transmission pathways, pathologic disease, immunization, and disease control can be affected by the genetic diversity of the parasite. Various studies indicated sheep strain G1 as the most common genotype throughout the world. This strain is commonly found in the liver and lung repeatedly causing echinococcosis in humans, sheep, and cattle. The present study was conducted to determine the genetic affinity between the protoscolex of E. granulosus in humans and sheep in East Azerbaijan province, Iran for the first time. A total of 120 hydatid cyst samples were collected, 60 of which were from people who referred to the hospitals of East Azerbaijan and 60 were from the sheep slaughtered in Tabriz slaughterhouse. Following DNA extraction, certain regions of the cox1 gene were amplified and evaluated by the polymerase chain reaction. The replicated parts in all isolates had the same size of 450 bp. Electrophoresis was followed by selecting a total of 60 suitable samples, including 30 human samples and 30 sheep samples and sending them for genome sequencing. The overlap of the samples was investigated using the BLAST software. The results of BLAST, sequencing, and overlap demonstrated a genetic linkage of approximately 91.76% between the protoscolex of E. granulosus in human and sheep.

In vitro effects of lonidamine and 6-aminonicotinamide against Echinococcus granulosussensu stricto and Echinococcus multilocularis.

Echinococcosis is a zoonotic disease caused by cestode species of the genus Echinococcus, which demonstrates considerable medical and veterinary concerns. The development of novel drugs for echinococcosis treatment is urgently needed. In this study, we demonstrated that lonidamine (LND) and 6-aminonicotinamide (6-AN) exhibited considerable in vitro effects against both larval- and adult-stage of E. granulosussensu stricto (s. s.) and E. multilocularis. The combination of LND and 6-AN exhibited a significantly higher activity than the single drug treatment. These results highlight the therapeutic potential of LND, 6-AN and the combination of LND and 6-AN for the treatment of echinococcosis.

The High Potential of Ozone Gas to Inactivate Echinococcus granulosus Protoscoleces During Hydatid Cyst Surgery.

BACKGROUND: In medicine, ozone therapy is effectively used in a broad spectrum of diseases. Reviews have shown that ozone gas demonstrates potent antimicrobial effects against a wide range of pathogenic microorganisms, such as oral bacteria, fungi, viruses, and parasite even in resistant strains. The present investigation was designed to assess the protoscolicidal effects of ozone gas on hydatid cysts protoscoleces in vitro and in vivo. METHODS: Hydatid cyst protoscoleces were acquired from sheep livers that were slaughtered at Kerman slaughterhouse, Iran. The viability of protoscoleces was assessed by the eosin exclusion examination after exposure with ozone gas for 1 to 14 min in vitro and ex vivo. RESULTS: In this study, in vitro assay showed that ozone gas at the concentration of 20 mg/L killed 85 and 100% of hydatid cyst protoscoleces after 4 and 6 min of treatment, respectively. However, in the ex vivo analysis, a longer time was needed to confirm a potent protoscolicidal activity such that ozone gas after an exposure time of 12 min, 100% of the protoscoleces were killed within the hydatid cyst. CONCLUSION: In conclusion, the findings of the present study showed that ozone gas at low concentrations (20 mg/L) and short times (4-6 min) might be used as a novel protoscolicidal drug for use in hydatid cyst surgery. However, more clinical surveys are required to discover the precise biological activity of ozone gas in animal and human subjects.

First molecular identification of hydatid tapeworm Echinococcus granulosus sensu lato G6/G7 in Ecuador.

Echinococcosis is a zoonotic parasitic illness that can cause significant disabilities, and even death for sick people. The disease is caused by the larval stage of cestodes belonging to the Echinococcus genus. In this study, multiple hydatid cysts were excised from an infected porcine liver. The identification of the parasitic species was made by the morphometric assessment of rostellar hooks and molecular detection of ribosomal DNA extant in protoscoleces of the hydatid sand. Rostellar hooks presented an average length of 27.4 µm by optical microscopy. Parasite DNA were detected in samples of hydatid sediment and positive controls by polymerase chain reaction. In conclusion, Echinococcus granulosus was recognized in samples of porcine hydatid cysts by microscopic observation, and the E. granulosus sensu lato strain E. canadensis G6/G7 was identified by molecular assay.

Unraveling oxidative stress response in the cestode parasite Echinococcus granulosus.

Cystic hydatid disease (CHD) is a worldwide neglected zoonotic disease caused by Echinococcus granulosus. The parasite is well adapted to its host by producing protective molecules that modulate host immune response. An unexplored issue associated with the parasite's persistence in its host is how the organism can survive the oxidative stress resulting from parasite endogenous metabolism and host defenses. Here, we used hydrogen peroxide (H2O2) to induce oxidative stress in E. granulosus protoescoleces (PSCs) to identify molecular pathways and antioxidant responses during H2O2 exposure. Using proteomics, we identified 550 unique proteins; including 474 in H2O2-exposed PSCs (H-PSCs) samples and 515 in non-exposed PSCs (C-PSCs) samples. Larger amounts of antioxidant proteins, including GSTs and novel carbonyl detoxifying enzymes, such as aldo-keto reductase and carbonyl reductase, were detected after H2O2 exposure. Increased concentrations of caspase-3 and cathepsin-D proteases and components of the 26S proteasome were also detected in H-PSCs. Reduction of lamin-B and other caspase-substrate, such as filamin, in H-PSCs suggested that molecular events related to early apoptosis were also induced. We present data that describe proteins expressed in response to oxidative stress in a metazoan parasite, including novel antioxidant enzymes and targets with potential application to treatment and prevention of CHD.

Extracellular vesicles derived from Echinococcus granulosus hydatid cyst fluid from patients: isolation, characterization and evaluation of immunomodulatory functions on T cells.

Cystic echinococcosis is a chronic and complex zoonotic disease. The mechanisms underlying the parasite's establishment, growth and persistence are not completely understood, and are thought be modulated by a crosstalk through extracellular vesicles. Here, EVs were isolated from the hydatid cyst fluid of patients with cystic echinococcosis and protoscolex culture supernatant. Proteomic analysis of these EVs revealed several parasite- and human-derived proteins. Very few studies have performed proteomic analysis of EVs isolated from HCF and PCS. Our proteomic analysis of the EVs derived from HCF and PCS facilitated identification of 1175 proteins, wherein 1026 and 38 proteins were exclusively identified in the EVs derived from HCF (HCF-EVs) and PCS (PCS-EVs), respectively, and 111 proteins were shared in both. The results of co-culture of PCS-EVs with murine peripheral blood mononuclear cells showed that PCS-EVs significantly regulated T lymphocyte functions in a dose-dependent manner. Collectively, our results provide valuable information on parasite survival strategies and new insights into the role of these EVs in the establishment and persistence of hydatid cysts.

Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton.

Echinococcus granulosus is a cestode parasite whose cytoskeleton plasticity allows it to enter and develop inside its hosts, completing thus its life cycle. We focused our attention on F-actin organization and distribution in E. granulosus protoscoleces (PSC) in order to contribute to the knowledge of the parasite cytoskeleton. In particular, we addressed some aspects of F-actin rearrangements in PSC at different stages of the evagination/invagination process. The use of light microscopy allowed us to identify different PSC structures and phalloidin staining displayed a parasite's highly organized F-actin cytoskeleton. Suckers exhibit an important musculature composed of a set of radial fibers. At the rostellum, the F-actin filaments are arranged in a bulbar shape with perforations that appear to be the attachment places for the hooks. Also, "circular" structures of F-actin were identified, which remind the flame cells. Furthermore, parasite F-actin filaments, unevenly distributed, seem to have remained substantially unchanged during the evagination/invagination process. Finally, we showed that the scolex of an evaginated E. granulosus PSC reinvaginates in vitro without any treatment.

Prevalence estimation of Italian ovine cystic echinococcosis in slaughterhouses: A retrospective Bayesian data analysis, 2010-2015.

Cystic echinococcosis (CE) is a complex zoonosis with domestic and sylvatic life-cycles, involving different intermediate and definitive host species. Many previous studies have highlighted the lack of a surveillance system for CE, its persistence in Italy, and endemicity in several Italian regions. Because of the absence of a uniform surveillance program for both humans and animals, disease occurrence is widely underestimated. This study aimed to estimate the prevalence of ovine CE in Italy. Survey data on the prevalence of Echinococcus granulosus complex infections in Italian sheep farms from 2010 to 2015 were obtained in collaboration with Regional Veterinary Epidemiology Observatories (OEVRs). Bayesian analysis was performed to estimate the true CE farm prevalence. The prior true CE prevalence was estimated using data from Sardinia. Second, Bayesian modelling of the observed prevalence in different regions and the true prevalence estimation from the first step were used to ultimately estimate the prevalence of ovine CE in Italy. We obtained survey data from 10 OEVRs, covering 14 Italian regions. We observed that the risk of CE infection decreased over the years, and it was strictly correlated with the density of susceptible species. Using Sardinia as prior distribution, where the disease farm prevalence was approximately 19% (95% CI, 18.82-20.02), we estimated that the highest endemic CE farm prevalence was in Basilicata with a value of 12% (95% BCI: 7.49-18.9%) and in Piemonte 7.64%(95% BCI: 4.12-13.04%). Our results provide spatially relevant data crucial for guiding CE control in Italy. Precise information on disease occurrence location would aid in the identification of priority areas for disease control implementation by the authorities. The current underestimation of CE occurrence should urge the Italian and European governments to become aware of the public health importance of CE and implement targeted interventions for high-risk areas.

Calmodulin-specific small interfering RNA induces consistent expression suppression and morphological changes in Echinococcus granulosus.

Among parasitic helminths, biological features of Echinococcus granulosus have been a focus of particular interest in biology and medicine. The determinants and underlying molecular mechanisms of Echinococcus development in different host settings is largely unknown. The phenomenal bi-directional development of E. granulosus protoscoleces into multi-proglottid and/or microcysts, is a fascinating feature of the parasite cultivation. Calmodulin (CaM) is the major intracellular Ca2+ binding protein in plant and animal organisms. Many Ca2+-related processes in the physiology of eukaryotic organisms are CaM-dependent, however little is known on the role of CaM in platyhelminths growth and development. Small interfering (si) RNA-induced manipulations of the genes involving in the parasite development is an opportunity to explore novel approaches for cystic echinococcosis (CE) prevention and management. Regarding the fundamental role of CaM in cellular function of the parasites, in this study, we investigated the molecular and morphological changes induced by siRNA on CaM in different in vitro stages of E. granulosus. Three developmental stages of the tapeworm, protoscoleces, microcysts and strobilated worms, were cultivated in vitro in mono- and di-phasic media and three delivery methods, i.e. electroporation, soaking and electro-soaking, were used for RNA interference. The level of mRNA suppression as well as the phenotypic changes of the parasites were measured. Following RNA interference, EgCaM mRNA suppressions of 65-99% were recorded in different stages of the tapeworm as compared to untreated/unrelated siRNA controls. Lower viability, growth retardation, morphological abnormalities as well as EgCaM expression suppression were documented in the parasite implying potential of siRNA technology for the prevention and management of CE.