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.

Natural infection in Pampas fox (Lycalopex gymnocercus) by Lagochilascaris major Leiper, 1910 (Nematoda: Ascarididae) in Buenos Aires, Argentina.

Lagochilascariosis is an emerging parasitic disease limited to the American continent, caused by nematodes of the genus Lagochilascaris. Its life cycle is heteroxenous, involving natural definitive hosts (wild carnivores), accidental hosts (domestic carnivores and humans), and intermediate hosts (rodents). Here we report, for the first time, the occurrence of Lagochilascaris major in female of a Pampas fox (Lycalopex gymnocercus) which was found in the Mar Chiquita basin, Buenos Aires province, Argentina. At necropsy, multiple Lagochilascaris larvae and several adults (1 female and 5 male) were observed inserted in the mucosa of the trachea and a male was also collected in the stomach. The diagnosis was made by observing the different stages with optical microscopy, by scanning electron microscopy of adult males and coproparasitological analysis. Although cases of lagochilascariosis have been reported in wildlife in different countries of the American continent, in Argentina, cases are really rare and were only found in domestic cats. Graphical abstract ᅟLagochilascariosis is an emerging parasitic disease limited to the American continent. In Argentina, sporadic cases of Lagochilascariosis were reported in domestic cats but this work represents the first record in a wild carnivore (L. gymnocercus) naturally infected with L. major.

First morphological and molecular analysis of Eucoleus boehmi like eggs in dogs from Argentina.

The canid parasites Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi) parasitize the lower and the upper respiratory tract, respectively. Reports and descriptions of these nematodes are scarce in Argentina, possibly due to misdiagnosis of morphologically similar trichuroids eggs, and the lack of knowledge about the species of Eucoleus in this geographical area. Scanning electron microscopy is a useful tool for identification of E. boehmi eggs based on the characteristics of the shell structure which differentiate between species. Molecular analysis complements morphological identification. Until now, there are no studies based on the analysis of E. boehmi eggs in Argentina. The aim of the present work was to study by morphological, morphometric, and molecular analysis, eggs attributable to E. boehmi isolated from dogs naturally infected in Mar del Plata city, Argentina. Eggs isolated from two dog fecal samples were analyzed by light and scanning electron microscopy. A fragment of mitochondrial DNA (mtDNA) of the cytochrome c oxidase subunit I gene (cox1) from eggs was sequenced, and phylogenetic analysis was performed in this study. According to morphological results based on the wall surface ultrastructure, the eggs studied were assigned to E. boehmi. Molecular analysis supported the morphological identification. The divergence of 9-12% with the European isolated could suggest a new geographical genetic variation of E. boehmi, but also question the possible existence of cryptic species. This is the first characterization of E. boehmi eggs in dogs from Argentina.

Epidemiological studies on Echinococcus in Pampas fox (Lycalopex gymnocercus) and European hare (Lepus europaeus) in Buenos Aires province, Argentina.

In Argentina, hydatid disease caused by Echinococcus granulosus is widespread. The south of Buenos Aires province, Argentina, is one of the three regions where hydatidosis is endemic. Although domestic dogs and sheep are considered to be the main hosts for E. granulosus, the potential role of wildlife in the local transmission of E. granulosus has not been investigated. The aim of this study was to estimate the hydatidosis/echinococcosis prevalence in European hare (Lepus europaeus) and Pampas fox (Lycalopex gymnocercus), two abundant species with a strong predator-prey relationship in rural areas of Buenos Aires province using different diagnostic tests. A total of 61 fox intestines were examined, finding that 52 (85.2%) harbored at least one helminth species. However, no adult or immature form of Echinococcus sp. was found in the intestinal contents. Coproparasitological analysis and Copro-ELISA followed by Copro-PCR were used as supplementary diagnostic tests. Only one (1.7%) of 59 fecal samples was positive to Taeniidae eggs by coproparasitological analysis, but this same sample was negative by the Copro-ELISA test. The analysis by Copro-ELISA showed 6 of 57 (10.6%) positive samples, but the Copro-PCR tests carried out on these samples were negative to E. granulosus. A total of 6,808 lungs, 3,576 livers, and 3,542 hearts of hunted hares were examined and palpated, but no structure resembling hydatid cysts were detected. Our results suggest that hares and Pampas foxes are not currently important wild reservoirs of E. granulosus in the studied area.

Pampas fox (Lycalopex gymnocercus) of the Argentine Pampas as intermediate host for Neospora caninum.

The Pampas fox (Lycalopex gymnocercus) is the most abundant wild canid from South America. This wild canid inhabits grasslands, open woodlands, and areas highly modified by extensive ranching and agricultural activities. We aimed to evaluate Neospora caninum infection in tissues from the Pampas fox from Argentina. A total of 41 free-living Pampas foxes were sampled in rural areas located in the Humid Pampas region, Argentina. Brain tissue and different muscles were assessed by histologic and molecular methods. No N. caninum cysts were observed in brain and muscle tissue samples analyzed by histology and immunohistochemistry. Molecular N. caninum identification from brain tissue was based on amplification by PCR of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. The presence of N. caninum DNA was 74% (23/31) for the Nc-5 gene and was confirmed by a second ITS1 PCR in 55% (17/31) of the brain tested. Thirteen ITS1 consensus sequences were obtained, and all have a 99.58-100% similarity with N. caninum reference sequences. Only 4% (1/23) of muscles samples analyzed were positive for the Nc-5 gene of N. caninum. This study demonstrated a high prevalence of N. caninum DNA in brain from free-ranging Pampas fox of the Pampa Argentine, thus confirming that this wild canid is a wide distributed intermediate host.

Molecular characterization of Sarcocystis spp. in intestine mucosal scrapings and fecal samples of Pampas fox (Lycalopex gymnocercus).

Sarcocystis spp. are obligatory intracellular protozoan parasites which can infect humans and animals. Most of Sarcocystis species were identified based on the detection of muscle cysts in different intermediate hosts (IH). Regarding to natural infection in definitive host, there are few reports which have reached to determining species of Sarcocystis. The present work was aimed to studying the occurrence of Sarcocystis spp. (oocysts and sporocysts) in mucosal scrapings of small intestine and fecal samples of one the most abundant wild canids from South America, Lycalopex gymnocercus (Pampas fox), and to identify the Sarcocystis spp. using molecular tools. A total of 131 free-living L. gymnocercus were sampled in rural areas located in several departments from Buenos Aires province, Argentina. Fecal samples from all the animals and 33 small intestines were analyzed. Fecal and mucosal scrapings samples were analyzed by sugar flotation method and once oocysts or sporocysts were detected, sedimentation was performed and DNA extracted with a commercial kit. A PCR was conducted using primers targeting a fragment of the 18S rRNA gene and the amplicons were purified and sequenced. Of the total Pampas foxes analyzed, 23 (17.6%) had Sarcocystis spp. oocysts/sporocysts in fecal and/or mucosal samples. Sarcocystis spp. sporocysts were detected in 13.0% (17/131) of fecal samples and in 39.4% (13/33) of mucosal samples by the initial sugar flotation. Twenty one L. gymnocercus samples were processed by DNA extraction and PCR. Molecular identification of Sarcocystis spp. infection was successfully achieved in 14 foxes and was distributed as follows: 4.6% S. cruzi (6/131), 3.8% Sarcocystis spp. using birds as IH (S. albifronsi and S. anasi among others, 5/131), 0.8% S. tenella (1/131) and 1.5% (2/131) with low homology (97%) with S. miescheriana. In one fecal sample with spherical oocysts, the sequencing results showed a 100% sequence identity with Hammondia heydorni. The results show that the mucosal scrapings are the eligible sample to identify prevalence and to proceed with species identification. Lycalopex gymnocercus is suggested as definitive host for S. cruzi, S. tenella and probably various Sarcocystis spp. using birds as intermediate hosts as well as for H. heydorni.

Reprint of "Survey and first molecular characterization of Echinococcus granulosus sensu stricto (G1) in Pampas fox (Lycalopex gymnocercus) in Buenos Aires province, Argentina".

Echinococcosis is a zoonosis caused by tapeworms of the genus Echinococcus. Echinococcus granulosus sensu lato (s. l.) has a world-wide distribution and its transmission is primarily maintained in a synanthropic cycle with dogs as definitive hosts and livestock species as intermediate hosts. However, many wild canids also function as definitive hosts for E. granulosus s. l. Echinococcosis in humans is mainly caused by E. granulosus sensu stricto (s. s.) G1 genotype. In the present work, we expanded the epidemiological study on echinococcosis reported cases in Pampas fox (Lycalopex gymnocercus) to provide a prevalence estimate for rural areas of southern Buenos Aires province, Argentina. Ninety-five whole intestines were analyzed using the sedimentation and counting technique with a result of 83 foxes (87.37%) harboring at least one helminth species. E. granulosus s. l. adults were found in one Pampas fox (1.05%). These adult helminthes were E. granulosus s. s. (G1) according to the genotyping analysis of a 450-bp region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene.

Ancylostoma (Ancylostoma) buckleyi (Nematoda: Ancylostomatidae): new wild host and distribution expansion.

Here we report the occurrence of Ancylostoma (Ancylostoma) buckleyi (Le Roux and Biocca, 1957) (Nematoda: Ancylostomatidae) in the small intestine of Pampas foxes (Lycalopex gymnocercus) (Mammalia: Canidae). This fox is the most abundant native carnivore in southern South America, where it inhabits grasslands, open woodlands and areas highly modified by extensive ranching and agricultural activities. Material from 80 foxes in rural areas of southern Buenos Aires province, Argentina was examined. The intestinal tracts were carefully removed from each carcass and subsequently isolated by ligatures (pylorus and rectum). Examination of the intestinal content was performed using the sedimentation and counting technique. Four foxes (5%) were found to be parasitized with adult specimens of A. buckleyi. This is the first report of Ancylostoma (A.) buckleyi in Argentina and adds L. gymnocercus as new host of this nematode species.

Survey and first molecular characterization of Echinococcus granulosus sensu stricto (G1) in Pampas fox (Lycalopex gymnocercus) in Buenos Aires province, Argentina.

Echinococcosis is a zoonosis caused by tapeworms of the genus Echinococcus. Echinococcus granulosus sensu lato (s. l.) has a world-wide distribution and its transmission is primarily maintained in a synanthropic cycle with dogs as definitive hosts and livestock species as intermediate hosts. However, many wild canids also function as definitive hosts for E. granulosus s. l. Echinococcosis in humans is mainly caused by E. granulosus sensu stricto (s. s.) G1 genotype. In the present work, we expanded the epidemiological study on echinococcosis reported cases in Pampas fox (Lycalopex gymnocercus) to provide a prevalence estimate for rural areas of southern Buenos Aires province, Argentina. Ninety-five whole intestines were analyzed using the sedimentation and counting technique with a result of 83 foxes (87.37%) harboring at least one helminth species. E. granulosus s. l. adults were found in one Pampas fox (1.05%). These adult helminthes were E. granulosus s. s. (G1) according to the genotyping analysis of a 450-bp region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene.

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.

The Pampas fox (Lycalopex gymnocercus) as new definitive host for Spirometra erinacei (Cestoda: Diphyllobothriidae).

Spirometra erinacei, Faust, Campbell and Kellogg, 1929, is a pseudophyllidean cestode of the family Diphyllobothriidae. The genus Spirometra is cosmopolitan and these parasites infect carnivores, specially felids and canids. In Argentina, S. erinacei and S. mansonoides have been reported sporadically only in domestic definitive hosts. The Pampas fox, Lycalopex gymnocercus, is the most abundant native carnivore in southern South America, where it inhabits grasslands and open woodlands and areas highly modified by extensive ranching and agricultural activities. This report describes the first finding of S. erinacei infecting Pampas fox, and provides an estimate prevalence of this cestode in rural areas of southern Buenos Aires province, Argentina based on 78 complete Pampas fox intestine samples analysis. This study found a 15.4% of prevalence of S. erinacei in small intestine (adult stage) and a 21.8% in fecal samples (egg stage). In the present work, the first case of S. erinacei in a wild definitive host from Argentina was reported expanding the list of definitive hosts of S. erinacei in South America.

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.

Ancylostoma (Ancylostoma) buckleyi (Nematoda: Ancylostomatidae): new wild host and distribution expansion / Ancylostoma (Ancylostoma) buckleyi (Nematoda: Ancylostomatidae): novo hospedeiro salvagem e expansão de distribuição

Abstract Here we report the occurrence of Ancylostoma (Ancylostoma) buckleyi (Le Roux and Biocca, 1957) (Nematoda: Ancylostomatidae) in the small intestine of Pampas foxes (Lycalopex gymnocercus) (Mammalia: Canidae). This fox is the most abundant native carnivore in southern South America, where it inhabits grasslands, open woodlands and areas highly modified by extensive ranching and agricultural activities. Material from 80 foxes in rural areas of southern Buenos Aires province, Argentina was examined. The intestinal tracts were carefully removed from each carcass and subsequently isolated by ligatures (pylorus and rectum). Examination of the intestinal content was performed using the sedimentation and counting technique. Four foxes (5%) were found to be parasitized with adult specimens of A. buckleyi. This is the first report of Ancylostoma (A.) buckleyi in Argentina and adds L. gymnocercus as new host of this nematode species.

Resumo O presente estudo relata a ocorrência de Ancylostoma (Ancylostoma) buckleyi (Le Roux and Biocca, 1957) (Nematoda: Ancylostomatidae) no intestino delgado do Graxaim do campo (Lycalopex gymnocercus) (Mammalia: Canidae). Essa raposa é o carnívoro nativo mais abundante no sul da América do Sul, onde habita nas pastagens, florestas abertas e áreas altamente modificadas pelas atividades pecuária extensiva e agrícola. Material de 80 raposas, em áreas rurais do sul da província de Buenos Aires, Argentina, foi examinado. Os tratos intestinais foram cuidadosamente removidos de cada carcaça e, posteriormente, isolados por ligaduras (piloro e reto). O exame do conteúdo intestinal foi realizado, utilizando-se a técnica de sedimentação e contagem. Quatro raposas (5%) foram encontradas parasitadas com espécimes adultos de A. buckleyi. O estudo registra, pela primeira vez, a ocorrência de Ancylostoma (A.) buckleyi na Argentina e adiciona L. gymnocercus como novo hospedeiro dessa espécie de nematoides.

Relevamiento de parásitos zoonóticos en materia fecal canina y su importancia para la salud de los niños / Coproparasitological canine survey in the Centro Municipal de Zoonosis and public squares

La ciudad de Mar del Plata presenta una población estimada de 100.000 canes. Esta situación genera alta contaminación del ambiente con materia fecal canina, incrementando la probabilidad de infección por parásitos zoonóticos para las personas y perros que concurren a los espacios públicos. El objetivo del presente trabajo fue analizar conjuntamente los resultados obtenidos a partir del análisis de materia fecal canina procedente de plazas públicas y del Centro Municipal de Zoonosis, para dimensionar el riesgo sanitario existente en el ambiente. Material y métodos. Entre septiembre de 2001 y marzo de 2002 se examinaron coproparasitológicamente 205 perros ingresados al Centro Municipal de Zoonosis y 288 muestras provenientes de 21 plazas de la ciudad. Las muestras se procesaron mediante técnica de flotación-sedimentación de Willis. Se calcularon las prevalencias de parásitos totales, los porcentajes por especie y los porcentajes de muestras monoparasitadas y poliparasitadas. Los resultados se compararon aplicando la prueba de x2. Resultados. Las especies identificadas en plazas fueron: uncinarias, Trichuris vulpis, Toxocara canis, coccidios y amebas. En el Centro Municipal de Zoonosis, además de éstas, se identificaron Capillaria aerophila y Dipylidium caninum. La prevalencia total de parásitos fue significativamente mayor en Centro Municipal de Zoonosis y también lo fue porcentaje de muestras poliparasitadas. Conclusiones. Los resultados de este estudio demuestran una alta prevalencia de enteroparásitos caninos de importancia en salud pública y veterinaria en los caninos de la ciudad, por lo que se hace necesario implementar campañas de salud pública para concientizar a la población y generar conductas de tenencia responsable de mascotas

Dominancia mensual de acaros oribatidos (acarina) en una zona endémica a la cestodiasis ovina / Monthly dominance of oribatid mites (acarina) in endemic zone of sheep cestodiasis

Se realizaron muestreos mensuales de suelos en dos establecimientos agropecuarios dedicados a la cría de ovinos de la raza Lincoln, en el Partido de Puan, provincia de Buenos Aires, Argentina. Se identificaron 17 especies de oribátidos pertenecientes a 9 superfamilias. Zigoribatula lata, Z. elongata y Ceratozetes sp. habían demostrado anteriormente su papel como hospedadores intermediarios de Moniezia expansa y Helictometra giardi (Cestoda Anoplocephalidae). En base a los datos aportados por los muestreos realizados se discute el posible papel de las especies de oribátidos halladas en la región como hospedadores intermediarios de anoplocefálidos de rumiantes domésticos