Evaluation of the Protoscolicidal Activity of Humulus lupulus Methanolic Extracts on Echinococcus granulosus Sensu Stricto.

The larval stage of the parasite Echinococcus granulosus sensu lato (s.l) is responsible for cystic echinococcosis (CE), a long-term infection affecting humans and animals worldwide, and constitutes a serious public health concern. If left untreated, CE can cause serious damage to multiple organs, especially the liver and lungs. Regarding the treatment, in the last few years, the use of pharmacological treatment has increased, suggesting that in the future, drug therapy may replace surgery for uncomplicated cysts. However, the only available anthelmintic drug to treat this infection is the albendazole, which has an efficacy that does not exceed 50%. On the basis of the above-mentioned evidence, new and improved alternative treatments are urgently needed. The use of natural products and their active fractions and components holds great promise as a valuable resource for the development of novel and effective therapies. Hop (Humulus lupulus L.) is a bittering agent in the brewing industry for which the sedative, digestive, anti-inflammatory, and antimicrobial effects have been reported. The purpose of this study was to assess the in vitro efficacy of methanolic extracts from the leaves of hop varieties against E. granulosus sensu stricto (s.s) protoscoleces. Varieties Mapuche and Victoria caused a stronger protoscolicidal effect compared to the Bullion, Cascade, and Traful varieties (P < 0.01), coinciding with their highest content of flavonoids, total polyphenols, and saponins. The viability of protoscoleces treated with the varieties Mapuche and Victoria decreased to approximately 50% at days 5 y 8, respectively, showing alterations such as soma contraction and impaired microtriches. After 18 days of treatment with both varieties, protoscoleces were completely altered both structurally and ultrastructurally. In conclusion, the methanolic extracts of the H. lupulus varieties Mapuche and Victoria demonstrated a marked in vitro effect against E. granulosus s.s. protoscoleces. The beer-making industry exclusively uses hop cones, leaving behind large amounts of hop leaves as an agricultural by-product that is not being utilized. On the basis of our study, we propose that hop leaves could also be used as a source of secondary metabolites with anthelmintic activity.

Anthelmintic Effect of Cannabidiol against Echinococcus granulosus sensu stricto.

Cystic echinococcosis is a global parasitic zoonosis caused by infection with the larval stage of Echinococcus granulosus sensu lato. Cystic echinococcosis affects more than 1 million people worldwide, causing important economic costs in terms of management and livestock associated losses. Albendazole is the main drug used in treating human cystic echinococcosis. In spite of this, its low aqueous solubility, poor absorption, and consequently erratic bioavailability are the cause of its chemotherapeutic failures. Based on the described problem, new treatment alternatives urgently need to be developed. The aim of the present research was to study the in vitro and in vivo efficacy of cannabidiol (CBD), the second most abundant component of the Cannabis sativa plant, was demonstrated against E. granulosus sensu stricto. CBD (50 µg/mL) caused a decrease in protoscoleces viability of 80 % after 24 h of treatment which was consistent with the observed tegumental alterations. Detachment of the germinal layer was observed in 50 ± 10% of cysts treated with 50 µg/mL of CBD during 24 h. In the clinical efficacy study, all treatments reduced the weight of cysts recovered from mice compared with the control group. However, this reduction was only significant with ABZ suspension and the CBD + ABZ combination. As we could observe by the SEM study, the co-administration of CBD with ABZ suspension caused greater ultrastructural alteration of the germinal layer in comparison with that provoked with the monotherapy. Further in vivo research will be conducted by changing the dose and frequency of CBD and CBD + ABZ treatments and new available CBD delivery systems will also be assayed to improve bioavailability in vivo.

Experimental treatment of cystic echinococcosis: Combination therapy with carvacrol and thymol versus albendazole.

Cystic echinococcosis is a worldwide zoonotic disease caused by Echinococcus granulosus sensu lato (s.l.), which produces serious health and economic problems. For human treatment, chemotherapy with albendazole (ABZ), a derivative of benzimidazoles, is widely used. However, due to its low efficacy and the lack of alternatives to ABZ, novel compounds are urgently needed. Aromatic plants exhibit powerful pharmacological activities, are accessible, have a relatively low cost, and have generally mild toxicities, making them an effective choice to traditional therapies. In particular, the pharmaceutical properties of aromatic plants are partially attributed to essential oils (EOs). The aim of the present study was to assess the in vitro and in vivo effects of the combined carvacrol and thymol against E. granulosus sensu stricto (s.s.). The greatest protoscolicidal effect was observed with the 9:1 and 5:5 (carvacrol:thymol) combinations which caused a marked decrease in viability after 6 days post-incubation, agreeing with the ultrastructural changes obtained. Permeation of the cysts and loss of turgidity was observed with the incubation with the different combinations of carvacrol:thymol. In the clinical efficacy study, the combination of thymol (40 mg/kg) and carvacrol (40 mg/kg) caused a tendency to diminish the weight of the cysts in comparison with the control group. On the other hand, the treatment of infected mice with ABZ, thymol or carvacrol, caused a significant decrease in the weight of the cysts. In conclusion, we here demonstrated the efficacy of different concentrations of combined carvacrol and thymol against E. granulosus s.s. protoscoleces and murine cysts, where short periods of treatment were sufficient to achieve a pharmacological effect. Moreover, we observed a reduction in the weight of the cysts in experimentally infected mice after treatment with carvacrol and thymol. The strategy used has an advantage over synthetic drugs because natural compounds are generally safe and non-toxic. Moreover, the combination of two drugs with different modes of action would cause a reduction in the doses and treatment times. Based on the promising results obtained in vitro, in the future, different doses of the combined drugs will be assayed in vivo to determine the potential of these compounds for the treatment of cystic echinococcosis.

Identification and characterization of sirtuin enzymes in cestodes and evaluation of sirtuin inhibitors as new cestocidal molecules.

Anti-parasitic treatment of neglected tropical diseases caused by cestodes such as echinococcosis and cysticercosis relies on a small number of approved anthelmintic drugs. Furthermore, the treatment is usually prolonged and often partially effective and not well tolerated by some patients. Therefore, the identification of novel drug targets and their associated compounds is critical. In this study, we identified and characterised sirtuin enzymes in cestodes and evaluated the cestocidal potential of sirtuin inhibitors as new cestocidal molecules. Sirtuins are a highly conserved family of nicotinamide-adenine dinucleotide-lysine deacylases involved in multiple cellular functions. Here, we described the full repertoire of sirtuin-encoding genes in several cestode species. We identified six sirtuin-encoding genes that were classified into sirtuins Class I (SIRT1, SIRT2, and SIRT3), Class III (SIRT5), and Class IV (SIRT6 and SIRT7). In Echinococcus spp., sirtuin genes showed transcriptional expression throughout several developmental stages, sirtuin 2 (SIRT2) being the most expressed. To evaluate the potential of sirtuin inhibitors as new cestocidal molecules, we determined the in vitro effect of several Class I sirtuin inhibitors by motility assay. Of those, the selective SIRT2 inhibitor Mz25 showed a strong cestocidal activity in Mesocestoides vogae (syn. Mesocestoides corti) tetrathyridia at various concentrations. The Mz25 cestocidal activity was time- and dose-dependent with a half-maximal inhibitory concentration value significantly lower than that of albendazole. Additionally, Mz25 induced extensive damage in the general morphology with marked alterations in the tegument and ultrastructural features. By homology modelling, we found that cestode SIRT2s showed a high conservation of the canonical sirtuin structure as well as in the residues related to Mz25 binding. Interestingly, some non-conservative mutations were found on the selectivity pocket (an Mz25-induced structural rearrangement on the active site), which represent a promising lead for developing selective cestode SIRT2 inhibitors derived from Mz25. Nevertheless, the Mz25 molecular target in M. vogae is unknown and remains to be determined. This report provides the basis for further studies of sirtuins to understand their roles in cestode biology and to develop selective sirtuin inhibitors to treat these neglected tropical diseases.

The combination of carvacrol and albendazole enhanced the efficacy of monotherapy in experimental alveolar echinococcosis.

Alveolar echinococcosis is a helminthic zoonosis caused by the larval stage of Echinococcus multilocularis. When surgical resection of the parasite is not feasible, pharmacological treatment with albendazole is the only option. Due to the difficulties in achieving the success of treatment, it is necessary to find new drugs to improve the treatment of this disease. In the present work, the efficacy of carvacrol alone or combined with albendazole was evaluated against E. multilocularis metacestodes. The association of carvacrol with albendazole produced a greater in vitro effect than the compounds incubated separately. The most effective treatment was the combination of 10 µg/ml of carvacrol and 1 µg/ml of albendazole. In the clinical efficacy study, treatment of infected mice with carvacrol (40 mg/kg) and albendazole (25 mg/kg) reduced the weight of metacestodes by 29 % and 50 %, respectively; while the combination of drugs had an efficacy of 83 %. These results coincided with the tissue damage observed at the ultrastructural level. In conclusion, carvacrol and albendazole combination enhanced the efficacy of monotherapy. This strategy would allow to improve the efficacy of the treatment without increasing the doses of albendazole or lengthen the treatment period, reducing the occurrence of adverse effects.

Development of an improved anesthesia protocol to increase CF1 mice survival in a portal vein infection with Echinococcus granulosus sensu lato protoscoleces.

In order to optimize the survival rate of animals, the purpose of this study was to evaluate an injectable anesthesia protocol for the development of a murine model of hepatic cystic echinococcosis in female CF-1 mice. Three protocols of injectable anesthesia were evaluated during the infection of mice with Echinococcus granulosus sensu lato protoscoleces via the portal vein. The use or not of pre-anesthesia [atropine (0.4 mg/kg) and tramadol (2 mg/kg)] and the incorporation or not of yohimbine (0.5 mg/kg) (a reverser of xylazine) in mice anesthetized with ketamine/xylazine 80/8 mg/kg were evaluated. Most mice treated only with ketamine/xylazine 80/8 mg/kg did not achieve a deep surgical anesthetic plane. All mice treated with pre-anesthetic drugs achieved a deep surgical anesthetic plane after the administration of the anesthetic cocktail. Pre-anesthetic drugs application significantly reduced time induction of animals compared with those that received only anesthetic cocktail. Recovery time was significantly faster in the group that received yohimbine. Mice underwent laparotomy that did not receive yohimbine after surgery had a survival rate of 67%, whereas in the group treated with yohimbine the survival was 100 %. We recommend the protocol that applied pre-anesthetic drugs + ketamine/xylazine 80/8 mg/kg + yohimbine, as safe and reliable for the portal vein infection of mice with protoscoleces of E. granulosus sensu lato.

The potential for histone deacetylase (HDAC) inhibitors as cestocidal drugs.

BACKGROUND: Echinococcosis and cysticercosis are neglected tropical diseases caused by cestode parasites (family Taeniidae). Not only there is a small number of approved anthelmintics for the treatment of these cestodiases, but also some of them are not highly effective against larval stages, such that identifying novel drug targets and their associated compounds is critical. Histone deacetylase (HDAC) enzymes are validated drug targets in cancers and other diseases, and have been gaining relevance for developing new potential anti-parasitic treatments in the last years. Here, we present the anthelmintic profile for a panel of recently developed HDAC inhibitors against the model cestode Mesocestoides vogae (syn. M. corti). METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic screening was performed on M. vogae by motility measurements and optical microscopic observations. Some HDAC inhibitors showed potent anthelmintic activities; three of them -entinostat, TH65, and TH92- had pronounced anthelmintic effects, reducing parasite viability by ~100% at concentrations of ≤ 20 µM. These compounds were selected for further characterization and showed anthelmintic effects in the micromolar range and in a time- and dose-dependent manner. Moreover, these compounds induced major alterations on the morphology and ultrastructural features of M. vogae. The potencies of these compounds were higher than albendazole and the anthelmintic effects were irreversible. Additionally, we evaluated pairwise drug combinations of these HDAC inhibitors and albendazole. The results suggested a positive interaction in the anthelmintic effect for individual pairs of compounds. Due to the maximum dose approved for entinostat, adjustments in the dose regime and/or combinations with currently-used anthelmintic drugs are needed, and the selectivity of TH65 and TH92 towards parasite targets should be assessed. CONCLUSION, SIGNIFICANCE: The results presented here suggest that HDAC inhibitors represent novel and potent drug candidates against cestodes and pave the way to understanding the roles of HDACs in these parasites.

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.

Albendazole solid dispersions against alveolar echinococcosis: a pharmacotechnical strategy to improve the efficacy of the drug.

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.

Do albendazole-loaded lipid nanocapsules enhance the bioavailability of albendazole in the brain of healthy mice?

Neurocysticercosis is a neglected tropical disease that affects the central nervous system and is the most common cause of human epilepsy acquired in developing countries. Therapeutic failures attributed to medical management of neurocysticercosis with albendazole (ABZ) have been primarily linked to the poor drug absorption rate resulting in low drug level in plasma and brain tissue. The aim of the current work was to characterize and compare the brain pharmacokinetic behavior of ABZ formulated as a suspension or lipid nanocapsules (ABZ-LNCs) in healthy mice. The relative availability in brain tissue of the active metabolite ABZ sulphoxide increased 183% when ABZ was administered as LNCs, in relation to ABZ suspension. The parent drug was also detected for a short period of time. The bioavailability of ABZ in ABZ-LNCs treated mice increased more than 2 fold compared with ABZ suspension group. The enhanced drug brain exposure observed after administration of ABZ-LNCs to healthy mice has potential usefulness for the treatment of human neurocysticercosis.

Comparison of different staining methods for determination of viability on Mesocestoides vogae tetrathyridia.

Mesocestoides vogae is widely employed as a model for studying the biology, differentiation, and experimental chemotherapy of cestodes. Currently, there are few techniques to measure the viability of M. vogae metacestodes during pharmacological experiments. The aim of the present work was to evaluate and compare different staining techniques to determine objectively the viability of M. vogae tetrathyridia. Eosin (0.05% w/v), methylene blue (0.01% w/v), propidium iodide (PI, 2 µg/ml), and fluorescein diacetate (FDA, 0.5 µg/ml) solutions were tested against live, heat-killed (cultivated at 65 °C for 2 h) and thymol-treated tetrathyridia (50 and 250 µg/ml). Parasites were counted under a dissecting microscope or a fluorescence compound microscope, as appropriate. Studies by scanning electron microscope were performed to compare the ultrastructural damage with the viability of parasites. After comparing the performance of different dyes, we chose the eosin staining technique because its simplicity, rapidity, sensitivity, low cost and fidelity.

In vivo activity of albendazole in combination with thymol against Echinococcus multilocularis.

Human alveolar echinococcosis (AE) is caused by the fox tapeworm Echinococcus multilocularis and is usually lethal if left untreated. The current strategy for treating human AE is surgical resection of the parasite mass complemented by chemotherapy with benzimidazole compounds. However, reliable chemotherapeutic alternatives have not yet been developed stimulating the research of new treatment strategies such as the use of medicinal plants. The aim of the current study was to investigate the efficacy of the combination albendazole (ABZ)+thymol on mice infected with E. multilocularis metacestodes. For this purpose, mice infected with parasite material were treated daily for 20 days with ABZ (5 mg/kg), thymol (40 mg/kg) or ABZ (5 mg/kg)+thymol (40 mg/kg) or left untreated as controls. After mice were euthanized, cysts were removed from the peritoneal cavity and the treatment efficacy was evaluated by the mean cysts weight, viability of protoscoleces and ultrastructural changes of cysts and protoscoleces. The application of thymol or the combination of ABZ+thymol resulted in a significant reduction of the cysts weight compared to untreated mice. We also found that although ABZ and thymol had a scolicidal effect, the combination of the two compounds had a considerably stronger effect showing a reduction in the protoscoleces viability of 62%. These results were also corroborated by optical microscopy, SEM and TEM. Protoscoleces recovered from ABZ or thymol treated mice showed alterations as contraction of the soma region, rostellar disorganization and presence of blebs in the tegument. However both drugs when combined lead to a total loss of the typical morphology of protoscoleces. All cysts removed from control mice appeared intact and no change in ultrastructure was detected. In contrast, cysts developed in mice treated with ABZ revealed changes in the germinal layer as reduction in cell number, while the treatment with thymol or the ABZ+thymol combination predominantly showed presence of cell debris. On the other hand, no differences were found in alkaline phosphatase (AP), glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activities between control and treated mice, indicating the lack of toxicity of the different drug treatments during the experiment. Because combined ABZ+thymol treatment exhibited higher treatment efficiency compared with the drugs applied separately against murine experimental alveolar echinococcosis, we propose it would be a useful option for the treatment of human AE.

Effect of Different Terpene-Containing Essential Oils on the Proliferation of Echinococcus granulosus Larval Cells.

Human cystic echinococcosis remains a major public health problem on several countries and the treatment strategies are not solved. The aim of the present work was to determine the in vitro effect of thymol and Mentha piperita, M. pulegium, and Rosmarinus officinalis essential oils on the proliferation of E. granulosus larval cells. Isolated cells and cellular aggregates were obtained from hydatid cyst's germinal layer and exposed to 1, 5, and 10 µg/ml of thymol and the different essential oils for 7 days. Drug effect was evaluated using test viability and scanning electron microscopy. Control cell culture viability was 2.1 x 10(6) (100%) after 7 days of incubation. At day 7, thymol 5 µg/ml caused a reduction in cell viability of 63% and the essential oils of M. piperita 10 µg/ml, M. pulegium 10 µg/ml, and R. officinalis 10 µg/ml produced a reduction in the viability of 77, 82, and 71%, respectively. Moreover essential oils caused reduction in cell number, collapsed cells, and loss of normal tridimensional composition of the aggregates. Due to the inhibitory effect caused by essential oils on E. granulosus cells we suggested that it would be an effective means for suppression of larval growth.

In vitro and in vivo effects of tamoxifen against larval stage Echinococcus granulosus.

Cystic echinococcosis is a zoonotic infection caused by the larval stage of the cestode Echinococcus granulosus. Chemotherapy currently employs benzimidazoles; however, 40% of cases do not respond favorably. With regard to these difficulties, novel therapeutic tools are needed to optimize treatment in humans. The aim of this work was to explore the in vitro and in vivo effects of tamoxifen (TAM) against E. granulosus. In addition, possible mechanisms for the susceptibility of TAM are discussed in relation to calcium homeostasis, P-glycoprotein inhibition, and antagonist effects on a putative steroid receptor. After 24 h of treatment, TAM, at a low micromolar concentration range (10 to 50 µM), inhibited the survival of E. granulosus protoscoleces and metacestodes. Moreover, we demonstrated the chemotherapeutic and chemopreventive pharmacological effects of the drug. At a dose rate of 20 mg/kg of body weight, TAM induced protection against the infection in mice. In the clinical efficacy studies, a reduction in cyst weight was observed after the administration of 20 mg/kg in mice with cysts developed during 3 or 6 months, compared to that of those collected from control mice. Since the collateral effects of high TAM doses have been largely documented in clinical trials, the use of low doses of this drug as a short-term therapy may be a novel alternative approach for human cystic echinococcosis treatment.

Enhanced chemoprophylactic and clinical efficacy of albendazole formulated as solid dispersions in experimental cystic echinococcosis.

Cystic echinococcosis is a chronic, complex, and still neglected disease. Although albendazole has demonstrated efficacy, only about one-third of patients experience complete remission or cure and 30-50% of treated patients develop some evidence of a therapeutic response. Different strategies have been developed in order to improve the albendazole water solubility and dissolution rate. The aim of the current work was to investigate the chemoprophylactic and clinical efficacy of an albendazole:poloxamer 188 solid dispersion formulation on mice infected with Echinococcus granulosus metacestodes. Albendazole formulated as solid dispersion had greater chemoprophylactic and clinical efficacy than albendazole alone. The improved in therapeutic efficacy could be a consequence of the increase in the systemic availability of albendazole sulfoxide. The work reported here demonstrates that in vivo treatment with albendazole:poloxamer 188 impairs the development of the hydatid cysts. This new pharmacotechnically based strategy could be a suitable alternative for treating cystic echinococcosis in humans.

Development of a cell line from Echinococcus granulosus germinal layer.

In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

Echinococcus granulosus genotype G1 dominated in cattle and sheep during 2003-2006 in Buenos Aires province, an endemic area for cystic echinococcosis in Argentina.

Cystic echinococcosis (hydatidosis) is a severe and widespread disease, caused by the larval stage of the tapeworm Echinococcus granulosus; it affects large numbers of humans and farm animals annually, causing serious health and economic problems. Molecular studies have identified large genetic variation within the E. granulosus complex, with various hosts displaying different susceptibility to different genotypes. For the effective management of the disease, one of the most pressing tasks is to combine epidemiological and genetic data to better understand the role of different hosts and genotypes in the transmission of the parasite. The aim of the present study was to describe the epidemiology of cystic echinococcosis in cattle and sheep, and to characterise the genotypes of E. granulosus present in these farm animals. The study was carried out in the Pampa region of Argentina, with a particular focus on Buenos Aires province, where cystic echinococcosis represents an important human and veterinary health problem. Among 513 cattle and 792 sheep, 11.9% and 4.0%, respectively, were infected with E. granulosus. Genetic characterisation of 42 isolates from cattle and 34 isolates from sheep was carried out by sequencing mitochondrial cox1 and nad1 genes. The vast majority of isolates were identified as genotype G1, except for a single sheep isolate determined as genotype G2, and a single cattle isolate that corresponded to genotype G5. Genotype G1 has previously been found to be the most infectious genotype to humans. As G1 was also the genotype principally responsible for cystic echinococcosis in Buenos Aires province, these results have important implications for developing effective disease control programmes to improve human and animal healthcare in this region.

Anthelmintic effect of Mentha spp. essential oils on Echinococcus granulosus protoscoleces and metacestodes.

The aim of the present work was to determine the in vitro effect of Mentha piperita and Mentha pulegium essential oils against Echinococcus granulosus and to compare the effectiveness of both oils according to the exposure time and concentration. Although both treatments had a protoscolicidal effect, M. pulegium had a considerably stronger effect than M. piperita. Essential oil of M. pulegium produced dose- and time-dependent effects. Maximal protoscolicidal effect was observed after 12 days of incubation and reached 0% after 18 days. This lack of viability was proved during the determination of infectivity into mice. Essential oil of M. piperita produced only a time-dependent effect. At 24 days p.i., the viability of protoscoleces decreased to approximately 50%. Scanning and transmission electron microscopy (SEM and TEM) demonstrated the drug-induced ultrastructural damage. On the other hand, a loss of turgidity was detected in all M. pulegium-treated cysts respective of the drug concentration. There was a correlation between the intensity of damage and the concentration of the essential oil assayed. Studies by SEM revealed that the germinal layer of treated cysts lost the feature multicellular structure. M. pulegium essential oil showed piperitone oxide as main compound in their composition, and we suggest that this component could be responsible of the markedly anthelmintic effect detected. Our data suggest that essential oils of Mentha spp. can be a promising source of potential protoscolicidal agents. The isolation of active anthelmintic constituents is in progress and may lead to the discovery of compounds with improved therapeutic value.

Primary cell culture of Echinococcus granulosus developed from the cystic germinal layer: biological and functional characterization.

Cell cultures of parasitic helminths are an invaluable tool for investigations of basic biological processes, as well as for development of improved chemotherapeutic agents and molecular interactions between host and parasite. We carried out a simple and efficient methodology to isolate Echinococcus granulosus germinal cells which were maintained for at least 4 months while cultivated in the presence of reducing agents and hormones. Microscopic analysis of the primary cell culture revealed the presence of cells with similar Echinococcus germinal cell morphology and behaviour. Population doubling time was estimated at 48 h, showing a rapid division rate. To discard possible host contamination, the specificity of the primary culture was tested by nested PCR, analyzing mdh gene expression and obtaining only one product with the expected size. We also studied the expression of specific E. granulosus proteins in primary cell culture. The novel and systematized method described here constitutes a powerful tool for investigations in cystic echinococcosis on biochemical and biological aspects related to the life cycle of the parasite and mechanisms of host-parasite interactions. This method also constitutes a powerful tool for the design of more efficient therapeutic alternatives.

Flubendazole interferes with a wide spectrum of cell homeostatic mechanisms in Echinococcus granulosus protoscoleces.

The problem of chemotherapeutic treatments for human echinococcosis has not been completely solved. The benzimidazole-methylcarbamates (BZD), broad-spectrum antihelminthic agents, such as mebendazole and albendazole are the only drugs licensed for treatment of hydatid cysts. These drugs bind directly to beta-tubulin causing the disruption of microtubule-based processes in helminths. However, the molecular bases of their multiple biological activities are poorly understood. Recently, the effect of halogenated derivative flubendazole (FLBZ), against E. granulosus larvae has been conclusively demonstrated. The comparative effectiveness of FLBZ, among other BZDs, was shown by means of vitality tests and time of appearance of morphological damage of larvae. In the present study, we examined biochemical and molecular changes on protoscoleces treated with FLBZ. We show that FLBZ induces: 1) an increase in cytosolic free calcium, 2) a decrease in tubulin transcripts, 3) a reduction of mMDH expression and 4) a significant decrease in glycogen levels. These results are consistent with the existence of multiple targets for FLBZ, such as calcium signaling and energy metabolism, and contribute to the understanding of the pharmaceutical properties of FLBZ.