Antiparasitic Effects of Asteraceae Species Extracts on Echinococcus granulosus s.s.

Cystic echinococcosis is a zoonotic disease caused by the parasite Echinococcus granulosus sensu lato (s.l.), which is worldwide distributed and causes long-lasting infections in animals and humans. The existing treatment is limited to the use of benzimidazoles, mainly albendazole (ABZ). However, it has unwanted side effects and its efficacy is about 50%. The Asteraceae family includes plants that have therapeutic applications (medicinal species) and has an important role in new drug development. The species belonging to a different genus of this family show a wide range of anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, and antiparasitic activities, among others. The aim of the present study was to evaluate the in vitro efficacy of extracts of four Asteraceae species against protoscoleces of E. granulosus sensu stricto (s.s.). On the other hand, the Stevia aristata extract was assessed on the murine cyst of E. granulosus (s.s.) and the efficacy of S. aristata extract was investigated in a murine model of CE. Stevia satureiifolia, S. aristata, Grindelia pulchella, and G. chiloensis extracts at 100 µg/mL caused a decrease in protoscoleces viability; however, S. aristata extract produced the greatest in vitro protoscolicidal effect. After 20 days of treatment with the highest concentration (100 µg/mL) of S. aristata extract, protoscoleces viability decreased to 0%. The tegumental changes observed by scanning electron microscopy were consistent with the reduction in vitality. The collapse of the germinal layer was registered in 60 ± 5.8% and 83.3 ± 12.0% of cysts treated during 4 days with 50 and 100 µg/ml, respectively. The half maximal effective concentration (EC50) value of the S. aristata extract against E. granulosus (s.s.) cysts was 47.86 µg/mL (96 h). The dosage of infected animals with the 50 mg kg-1 dose of S. aristata extract resulted in a significant reduction in cyst weight in comparison with the control group. In conclusion, S. aristata extract was demonstrated to exert a marked effect, both in vitro and in the murine model.

Anthelmintic activity of Stevia multiaristata extract against Echinococcus granulosus sensu stricto.

Cystic echinococcosis is a zoonotic disease caused by the larval stage of the parasite Echinococcus granulosus sensu lato. The available anti-parasitic treatment is mostly limited to a continuous administration of albendazole. However, due to its numerous side-effects and efficacy of around 50%, there is a need to find new drugs to improve the treatment for this disease. In the current study, the in vitro and in vivo efficacy of a Stevia multiaristata extract against E. granulosus sensu stricto (s.s.) was demonstrated. Stevia multiaristata extract (100 and 50 µg mL−1) caused a quick viability decrease on protoscoleces which was consistent with the observed tegumental alterations. Loss of turgidity was detected in 95 ± 3.4% of cysts incubated with S. multiaristata extract during 2 days (100 µg mL−1) and the collapse of the germinal layer was observed in 60 ± 9.3% of cysts treated with 100 µg mL−1 of the S. multiaristata extract during 4 days. The half maximal effective concentration value was 69.6 µg mL−1 and the selectivity index for E. granulosus s.s. cysts was 1.9. In this clinical efficacy study, the treatment of infected mice with the S. multiaristata extract (50 mg kg−1) caused a significant decrease in the weight of the cysts compared with the control group. These results coincided with the tissue damage observed in the cysts at the ultrastructural level. In conclusion, we observed high protoscolicidal and cysticidal effects, and significant reduction in the weight of the cysts in experimentally infected mice following treatment with the S. multiaristata extract.

Experimental murine model of neurocysticercosis: first report of cerebellum as a location for Mesocestoides corti tetrathyridia.

Neurocysticercosis is a parasitic disease caused by encysted larvae of Taenia solium in the human central nervous system. Cysts mainly affect the cerebral hemispheres, although they can also be found in ventricles, basal cisterns, and subarachnoid spaces, and rarely in the cerebellum. Given the impossibility of studying the disease in human patients, Cardona et al. (1999) developed a mouse model of neurocysticercosis, using Mesocestoides corti, a closely related cestode. This allows us to study the parasite-host relationship and the mechanisms involved in the disease, in order to improve the therapy. In this murine model of neurocysticercosis, the location of tetrathyridia in parenchyma, ventricles and meninges has already been reported. The aim of this work is to report the cerebellum as a new location for M. corti tetrathyridia in the murine model of neurocysticercosis. A murine model that reproduces the human pathology is essential to evaluate the symptomatology and response to drug treatment in experimentally infected mice.

Could beta-myrcene be an alternative to albendazole for the treatment of experimental cystic echinococcosis?

Echinococcus granulosus causes hydatidosis or cystic echinococcosis in humans and livestock. In humans, this disease can be managed with surgery, percutaneous treatment, chemotherapy and/or observation. The chemotherapeutic agents used and approved for treatment of hydatidosis are benzimidazoles. Because of the difficulties in achieving successful treatment, considerable efforts have been made to find new natural compounds against hydatid disease. Beta-myrcene is a monoterpene presented in the essential oils of different plants. It is the principal component of essential oil of Rosmarinus officinalis (rosemary). The goal of the present study was to evaluate the in vitro effects of beta-myrcene against germinal cells, protoscoleces and murine cyst of E. granulosus, as well also, investigate its chemoprophylactic activity in a murine model of cystic echinococcosis. For the in vitro assays, the parasites were incubated with beta-myrcene at 10, 5 and 1 µg/mL. The treatments were dose and time-dependent, and consistent with the observed morphological alterations. In the chemoprophylactic efficacy study, the effect of beta-myrcene was similar to albendazole, the reference drug for human echinococcosis treatment.

Chemoprophylactic and therapeutic efficacy of thymol in murine cystic echinococcosis.

Cystic echinococcosis is a zoonotic disease caused by the larval stage of the cestode Echinococcus granulosus. The drugs commonly used against cystic echinococcosis are benzimidazoles. Unfortunately, 20%-40% of cases do not respond favorably to such chemotherapy. Consequently, the search of new therapeutic alternatives such as the use of traditional medicinal plants has been increased. The aim of the current experimental work was to investigate the chemoprophylactic and clinical efficacy of thymol on mice infected with E. granulosus metacestodes. Thymol (40 mg/kg) was administered under two different therapeutic schemes: dosing every 24h over 20 days and treatment every 12h for 10 days. Thymol demonstrated efficacy against experimental murine cystic echinococcosis. The chemoprophylactic and therapeutic effects of thymol were comparable to that of albendazole. Due to the lack of toxicity observed in mice at the tested doses; we consider that thymol is a potential alternative to be applied for the treatment of human hydatid disease.

Efficacy of Essential Oils of Thymus vulgaris and Origanum vulgare on Echinococcus granulosus.

The aim of the present work was to determine the in vitro effect of T. vulgaris and O. vulgare essential oils against E. granulosus protoscoleces and cysts. Essential oils were added to the medium resulting in thymol final concentrations of 10 µg/mL. The essential oils had a time-dependent effect provoking the complete loss of protoscolex viability after 72 days of postincubation. The results were confirmed at the ultrastructure level. Loss of infectivity in protoscoleces incubated with O. vulgare after 60 days was observed. On the other hand, the weight of cysts recorded in mice inoculated with T. vulgaris treated protoscoleces was significantly lower than that obtained in control group. Gamma-glutamyl-transpeptidase activity was readily detected in the culture supernatant of protoscoleces treated either with the essential oils or thymol. T. vulgaris and O. vulgare essential oils and thymol can induce cell apoptosis of protoscoleces after short incubation times. The efficacy of T. vulgaris and O. vulgare essential oils was also demonstrated in vitro on E. granulosus murine cysts. Our data suggest that essential oils of T. vulgaris and O. vulgare have anthelmintic effect against protoscoleces and cysts of E. granulosus.

In Vitro Cestocidal Activity of Thymol on Mesocestoides corti Tetrathyridia and Adult Worms.

Nothing is known about the possible effect of thymol or other compounds of essential oils against the adult worms of cestodes. The aim of the present work was to determine in vitro cestodicidal activity of thymol against Mesocestoides corti adult worms. Moreover, the in vitro effect on tetrathyridia was also demonstrated. Tetrathyridia exposed to different concentrations of thymol showed a concentration and time-dependent effect. At lower concentrations, the main change observed was mainly in morphology, with larvae exhibiting an elongation of the body. When tetrathyridia were exposed to higher concentrations, increased surface alterations and damage were detected. The body appeared elongated and flattened, and a complete loss of morphology and microtriches was observed. Thymol was able to kill M. corti tetrathyridia, since following inoculation of treated parasites in mice no parasites could be recovered. The effect on M. corti adult worms was dose and time-dependent. Changes in motility coincide with the tissue damage were observed at the structural and ultrastructural level. Thymol caused severe damages to both developmental stages analyzed. Damages were more significant in fully segmented worms. The data reported in this paper demonstrate a clear in vitro effect of thymol against M. corti tetrathyridia and adult worms.

In vitro effect of 5-fluorouracil and paclitaxel on Echinococcus granulosus larvae and cells.

Human cystic echinococcosis is a zoonosis caused by the metacestode stage of the tapeworm Echinococcus granulosus. Although benzimidazole compounds such as albendazole and mebendazole have been the cornerstone of chemotherapy for the disease, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of 5-fluorouracil (5-FU) and paclitaxel (PTX) against E. granulosus germinal cells, protoscoleces and cysts. 5-FU or PTX inhibited the growth of E. granulosus cells in a time dependent manner. Although both treatments had a protoscolicidal effect, 5-FU had a considerably stronger effect than PTX. 5-FU produced a dose- and time-dependent effect, provoking the complete loss of viability after 24 days of incubation. Moreover, cysts did not develop following the inoculation of treated protoscoleces into mice. The loss of viability was slower in PTX treated protoscoleces, reaching to approximately 60% after 30 days. The results of the in vitro treatment with 5-FU and PTX were similar in secondary murine cysts. The employment of SEM and TEM allowed us to examine, at an ultrastructural level, the effects induced by 5-FU and PTX on E. granulosus germinal cells, protoscoleces and murine cysts. In conclusion, the data obtained clearly demonstrated that 5-FU and PTX at clinically achievable concentrations inhibit the survival of larval cells, protoscoleces and metacestodes. In vivo studies to test the antiparasitic activities of 5-FU and PTX are currently being undertaken on the murine model of cystic echinococcosis.

Echinococcus granulosus: biological comparison of cattle isolates from endemic regions of Argentina and Spain / Echinococcus granulosus: comparación biológica de aislados de bovinos de regiones endémicas de Argentina y España

In the present study we have compared cattle isolates of Echinococcus granulosus from Argentina and Spain. The aim was to compare and determine if there exist phenotypic and genetic differences within E. granulosus cattle isolates between an endemic area of Spain (where the disease is mainly restricted to a sheep-dog cycle) and an endemic area of Argentina (where cattle are the most abundant intermediate hosts). The Spanish samples were previously identified as G1 genotype. The Argentinean samples were also identified as G1, but some variants were found for the cytochrome c oxidase-1 (CO1) and NADH dehydrogenase-1 (ND1) mitochondrial genes. When comparing the cyst features and the morphology of the larval rostellar hooks in both regions, some differences were found. The morphometric analyses of the larval rostellar hooks showed the existence of two distinct clearly separated groups (one corresponding to the Argentinean samples and the other to the Spanish ones). In conclusion, there are some genetic and phenotypic differences within E. granulosus cattle isolates from Argentina and Spain. Probably these differences, more important from an epidemiological point of view, are related to different steps in the disease control in both countries. Further studies involving other epidemiological, morphometric and molecular data, including other types of livestock, would contribute to clarify and expand the present work.

El objetivo del presente trabajo fue determinar si existen diferencias fenotípicas y genéticas entre los aislados de Echinococcus granulosus de origen bovino provenientes de dos regiones geográficas donde la hidatidosis es endémica, una de España (donde predomina el ciclo perro-oveja) y una de Argentina (donde el bovino es el hospedador intermediario más importante). Las muestras españolas fueron previamente identificadas como pertenecientes al genotipo G1. Las muestras argentinas también correspondían al genotipo G1, pero entre ellas se registraron algunas microvariantes de los genes mitocondriales citocromo c oxidasa-1 (CO1) y NADH deshidrogenasa- 1 (ND1). La comparación de las características de los quistes y de la morfología de los ganchos rostelares del metacestode mostró ciertas diferencias. En conclusión, existen algunas diferencias genéticas y fenotípicas entre los aislados de E. granulosus de Argentina y España. Probablemente estas diferencias, más importantes desde el punto de vista epidemiológico, podrían estar relacionadas con diferentes etapas en los programas de control de la enfermedad en los dos países. Estudios adicionales que involucren datos epidemiológicos, morfométricos y moleculares provenientes de otros tipos de ganado contribuirán a clarificar y ampliar la información aportada por este trabajo.

Pediatric hydatidosis in the south-east of the Buenos Aires province, Argentina.

Hydatidosis is a zoonosis produced by the metacestode Echinococcus spp. The aims of this research are: to contribute to the knowledge of pediatric hydatidosis in the south-east of Buenos Aires province, to study its evolution from 1993 to 2002 at the Regional Maternity and Pediatric Hospital "Dr. Victorio Tetamanti", to determine the strains involved and to discuss the importance of the disease. The clinical records of diagnosed and/or operated patients were reviewed with regard to the hydatid disease. The strain was determined by using PCRs with Eg1 121a/122a primers. Forty-four cases were analyzed. Fifty nine point one per cent of the patients were boys. The mean age was 8 SD=3.8 years. Sixty one point four per cent had urban residence. Ultrasonography was used in 61% of the cases. The hepatic location was most frequently seen and the liver/lung ratio was 1.25. Ninety point nine per cent of patients received surgical treatment. Albendazole was used in 52% of cases. The average hospitalization time was 11 days. The G1/G2 strain group was determined. This report is the first one of its kind in the studied region. The permanence of hydatidosis in the region depends on the natural transmission of the parasite in the absence of control and prevention measures. The health authorities should implement strategies of prevention and control in the study area.

Pediatric hydatidosis in the south-east of the Buenos Aires province, Argentina / Hidatidosis pediátrica en el sudeste de la provincia de Buenos Aires, Argentina

Hydatidosis is a zoonosis produced by the metacestode Echinococcus spp. The aims of this research are: to contribute to the knowledge of pediatric hydatidosis in the south-east of Buenos Aires province, to study its evolution from 1993 to 2002 at the Regional Maternity and Pediatric Hospital "Dr. Victorio Tetamanti", to determine the strains involved and to discuss the importance of the disease. The clinical records of diagnosed and/or operated patients were reviewed with regard to the hydatid disease. The strain was determined by using PCRs with Eg1 121a/122a primers. Forty-four cases were analyzed. Fifty nine point one per cent of the patients were boys. The mean age was 8 SD=3.8 years. Sixty one point four per cent had urban residence. Ultrasonography was used in 61% of the cases. The hepatic location was most frequently seen and the liver/lung ratio was 1.25. Ninety point nine per cent of patients received surgical treatment. Albendazole was used in 52% of cases. The average hospitalization time was 11 days. The Gl/G2 strain group was determined. This report is the first one of its kind in the studied region. The permanence of hydatidosis in the region depends on the natural transmission of the parasite in the absence of control and prevention measures. The health authorities should implement strategies of prevention and control in the study area.

Los objetivos de este trabajo fueron: contribuir al conocimiento de la hidatidosis pediátrica en el sudeste de la provincia de Buenos Aires, estudiar su evolución desde 1993 hasta 2002, establecer la o las cepas involucradas y discutir la importancia de la enfermedad. Para ello se revisaron las historias clínicas de los pacientes pediátricos con diagnóstico de hidatidosis asistidos en el Hospital Interzonal Especializado Materno Infantil "Dr. Victorio Tetamanti" durante ese período. Se analizaron 44 casos, el 59,1% de ellos correspondió a varones. La media de edad fue de 8 años (SD=3,8 años) y el 61,4% de los niños afectados eran de residencia urbana. Se empleó ultrasonografía como método diagnóstico en el 61% de los casos. La localización hepática fue la más frecuente y la relación hígado/pulmón fue 1,25. El 90,9% recibió tratamiento quirúrgico. Se utilizó albendazol en el 52% de los pacientes. El tiempo de hospitalización tuvo una mediana de 11 días. Las cepas se determinaron mediante PCR con los cebadores Eg1 121a/122a. Se determinó la presencia de cepas del grupo G1/G2, dato informado por primera vez en humanos para la región de estudio. Se concluyó que la permanencia de la enfermedad en la región depende de la transmisión natural del parásito en ausencia de medidas de control y prevención. Por consiguiente, las autoridades de salud deberían implementar estrategias de prevención y control en dicha zona.

Echinococcus granulosus: biological comparison of cattle isolates from endemic regions of Argentina and Spain.

In the present study we have compared cattle isolates of Echinococcus granulosus from Argentina and Spain. The aim was to compare and determine if there exist phenotypic and genetic differences within E. granulosus cattle isolates between an endemic area of Spain (where the disease is mainly restricted to a sheep-dog cycle) and an endemic area of Argentina (where cattle are the most abundant intermediate hosts). The Spanish samples were previously identified as G1 genotype. The Argentinean samples were also identified as G1, but some variants were found for the cytochrome c oxidase-1 (CO1) and NADH dehydrogenase-1 (ND1) mitochondrial genes. When comparing the cyst features and the morphology of the larval rostellar hooks in both regions, some differences were found. The morphometric analyses of the larval rostellar hooks showed the existence of two distinct clearly separated groups (one corresponding to the Argentinean samples and the other to the Spanish ones). In conclusion, there are some genetic and phenotypic differences within E. granulosus cattle isolates from Argentina and Spain. Probably these differences, more important from an epidemiological point of view, are related to different steps in the disease control in both countries. Further studies involving other epidemiological, morphometric and molecular data, including other types of livestock, would contribute to clarify and expand the present work.

Strain characterization of Echinococcus granulosus protoscoleces of cattle origin using the in vitro vesicular development.

The aim of this work was to characterize the strain of protoscoleces of E. granulosus of cattle origin using the in vitro vesicular development. The in vitro development of these samples was compared to samples of sheep origin determined previously by genetic analyses as common sheep strain (G1). There were similarities between sheep and cattle samples not only in the time of microcysts formation, but also in the development process. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. In the sheep samples, microcysts were observed between 19 and 20 days. In the cattle samples, microcysts appeared between 20 and 23 days. The coincidence between the development times and physiological characteristics found in the present study may indicate that the parasites from cattle and sheep were of the same strain.

Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique.

The aim of this work was the achievement of microcysts formation from protoscoleces of E. granulosus of cattle origin using the in vitro vesicular culture technique. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. On day 20, some microcysts with a complete laminated layer were observed. By day 48, microcysts completely developed could be observed. This is the first study where microcysts formation was obtained using protoscoleces of E. granulosus of cattle origin.

Viscachataenia n. g., with the redescription of V. quadrata (von Linstow, 1904) n. comb. (Cestoda: Anoplocephalidae) in Lagidium viscacia (Rodentia: Chinchillidae) from Argentina.

A new genus, Viscachataenia, is erected to accommodate the anoplocephalid cestode Cittotaenia quadrata von Linstow, 1904, based on a redescription of the species from material collected from the chinchillid rodent Lagidium viscacia in Argentina. The new genus is characterised by paired genitalia, a reticulate uterus and the vagina entering the genital atrium anterior to the cirrus-sac. Viscachataenia therefore has similarities with Monoecocestus Beddard, 1914, a genus which is common in South American rodents but which has a single set of genitalia in each segment. Cittotaenia viscaciae (Spasskii, 1951) and Bertiella findlayi Mazza, Parodi & Fiora, 1932, also from viscachas, are considered synonyms of V. quadrata.