Genotype and Phenotype of Echinococcus granulosus Derived from Wild Sheep (Ovis orientalis) in Iran.

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.

The optimum cut-off value to differentiate Echinococcus granulosus sensu stricto from other species of E. granulosus sensu lato using larval rostellar hook morphometry.

Cystic echinococcosis caused by Echinococcus granulosus sensu lato is one of the most important helminth zoonoses in the world; it affects both humans and livestock. The disease is endemic in Argentina and highly endemic in the province of Neuquén. Considerable genetic and phenotypic variation has been demonstrated in E. granulosus, and ten different genotypes (G1-G10) have been identified using molecular tools. Echinococcus granulosus sensu lato may be considered a species complex, comprised of E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-G10). In endemic areas, the characterization of cystic echinococcosis molecular epidemiology is important in order to apply adequate control strategies. A cut-off value for larval large hook total length to distinguish E. granulosus sensu stricto isolates from those produced by other species of the complex was defined for the first time. Overall, 1780 larval hooks of 36 isolates obtained from sheep (n= 11, G1), goats (n= 10, G6), cattle (n= 5, G6) and pigs (n= 10, G7) were analysed. Validation against molecular genotyping as gold standard was carried out using the receiver operating characteristic (ROC) curve analysis. The optimum cut-off value was defined as 26.5 µm. The proposed method showed high sensitivity (97.8%) and specificity (91.1%). Since in most endemic regions the molecular epidemiology of echinococcosis includes the coexistence of the widely distributed E. granulosus sensu stricto G1 strain and other species of the complex, this technique could be useful as a quick and economical tool for epidemiological and surveillance field studies, when fertile cysts are present.

In vitro protoscolicidal effects of high-intensity focused ultrasound enhanced by a superabsorbent polymer.

Echinococcus granulosus, the etiologic agent of cystic echinococcosis in humans and other animal hosts, is distributed worldwide. Echinococcosis is an increasing public health and socioeconomic concern. The present work evaluated whether or not a superabsorbent polymer (SAP) could enhance the damage efficacy of high-intensity focused ultrasound (HIFU) on the viability of E. granulosus protoscolices in vitro. HIFU of 100 W acoustic power and 0.01 g superabsorbent polymer were used to treat 5,000 protoscolices in 2-ml protoscolices suspension. After different HIFU exposure time (5, 10, 20, 30, 40, 50, and 60 s, respectively), the temperature of protoscolices suspension was taken, and the treated protoscolices were stained by trypan-blue exclusion assay, and their structures were observed by light microscopy. To better understand the biological mechanisms responsible for the deaths of protoscolices, the activity of succinate dehydrogenase (SDH) of the protoscolices treated with HIFU was examined. The temperature of protoscolices suspension treated with HIFU rose slowly, and the death rate of protoscolices was 73.7 % in the group of HIFU treatment time of 40 s; however, with the same HIFU treatment time of 40 s, the temperature of protoscolices suspension in the group of HIFU combined with SAP rose quickly, and the death rate of protoscolices was 100 %. The same protoscolicidal effect (100 %) of HIFU of 100 W acoustic power combined with SAP was also observed in the groups of HIFU treatment time of 50 and 60 s, respectively. The dead protoscolices were stained to blue, shrunken and black calcareous corpuscles, and disordered and decreasing hooks though of intact membrane, as well as some protoscolices lost hooks and tore open on membrane were observed. In the group of HIFU combined with SAP, it was found that the superabsorbent polymer was surrounded by the spoiled, and the destruction of protoscolices was much stronger than in the group of HIFU. The dead protoscolices exhibited the reduction or absence of SDH staining intensity in the parenchymal cell and calcareous corpuscles after HIFU irradiation, where a large number of necrotic cells were evident. These results suggested that HIFU could induce the damage and loss of viability of protoscolices; SAP could enhance the HIFU energy focused and cause more severe destruction efficacy on protoscolices; and mitochondrial energetic function is involved in the regulation of cell-death pathways of protoscolices.

The destructive effects of high-intensity focused ultrasound on hydatid cysts enhanced by ultrasound contrast agent and superabsorbent polymer alone or in combination.

Cystic echinococcosis, caused by the metacestode stage of Echincoccus granulosus, remains endemic in many regions around the world. The present work evaluated whether or not a superabsorbent polymer (SAP) and ultrasound contrast agent (UCA) alone or in combination could enhance damage efficacy of high-intensity focused ultrasound (HIFU) on hydatid cysts in vitro. HIFU of 100 W acoustic power, with the aid of 0.1 ml UCA and 0.1 g SAP alone or in combination, was used to ablate hydatid cysts in vitro. The comparison of ultrasound image for each layer of hydatid cyst before and after HIFU ablation was made immediately, and the protoscolices of the cysts were stained by eosin exclusion assay, and the structures of protoscolices were observed by light microscopy. To understand the destructive effects of HIFU, the pathological changes in cyst walls of hydatid cyst ablated with HIFU were examined. The results demonstrated that HIFU had some lethal effect on hydatid cysts: echo enhancement of ultrasound image, increase of mortality rate of protoscolices, serious structural damage of protoscolices, and complete destruction or even disappearance of laminated layer and germinal layer was observed in the group of HIFU combined with UCA and SAP alone or in combination. It was found that the destructive effect of HIFU aided with a combination of UCA and SAP to hydatid cysts was more effective than that of HIFU just aided with UCA or SAP alone. These results suggested that UCA and SAP might be used as a HIFU enhancing agent to improve the efficacy of HIFU ablation to hydatid cysts, which could be a possible therapeutic option for cystic echinococcosis.

Morphological characteristics of Echinococcus granulosus derived from buffalo in Iran.

Cystic echinococcosis is a significant parasitic disease in Iran, where a variety of animals act as intermediate hosts. In this study, 25 isolates of Echinococcus granulosus obtained from water buffalo from various parts of Iran were characterized on the basis of the morphology of the metacestode and the adult worm. The characteristics of protoscoleces from the different studied areas were nearly similar. They showed 2 rows of alternating large and small hooks and their shapes were smooth in outline. In contrast to the protoscoleces, the adult rostellar hooks showed a rough outline. The results showed that the total length, the blade lengths of the large and small hooks and the number of hooks are almost similar to those isolated from sheep but significantly different from those isolated from camels. The growth rates of adult E. granulosus (total worm length, segmentation and maturation) of buffalo origin, at 35 and 41 days post-infection of dogs, were nearly comparable to the common sheep strain. The form of the strobila and the morphology of the reproductive system were also similar to those of sheep origin. This suggests that the common sheep strain (G1) of E. granulosus may also use buffaloes as its intermediate host.

Rostellar hook morphology of larval Echinococcus granulosus isolates from the Indian buffalo and Iranian sheep, cattle and camel.

Isolates of Echinococcus granulosus from the Indian buffalo and Iranian sheep, cattle and camels were characterized on the basis of rostellar hook morphology of the protoscolices. Results obtained indicated phenotypic polymorphism among parasites isolated from different host species. Isolates from buffalo are morphologically quite different from those of the more common sheep and cattle isolates and may represent a different strain, adapted to buffalo. In the Sari region of northern Iran, two morphologically distinct forms of E. granulosus, one in sheep and one in camels, were identified. Total length and handle length of both large and small hooks were considered the most variable characteristics which could be used not only for differentiating parasite isolates from different host species but also the origin of infection in the definitive host. We therefore suggest that larval hook morphology may be considered as a valid criterion for the identification of E. granulosus strains in Iran.

Comparison of molecular and morphological characterization and haplotype analysis of cattle and sheep isolates of cystic echinococcosis.

Cystic echinococcosis is a zoonotic disease and the second most common foodborne parasitic infection worldwide. The aim of the present study was to investigate genetic variations in G1 and G3 genotypes of Echinococcus granulosus sensu stricto and determine the morphological differences between two genotypes. In total, 119 cystic samples were collected from 48 cattle and 71 sheep in slaughterhouses in four cities in three geographical regions of Turkey regions (Ankara, Central Anatolio region; Ordu, Black Sea region; and Adana, Mersin, Mediterranean region). For molecular characterization of the G1 and G3 genotypes, two gene regions (the complete mt-cox1 gene sequence and partial mt-nad5 gene sequence) were amplified. Haplotype analysis was conducted to determine the nucleotide differences between the complete sequences of the mt-cox1 gene for 47 samples. In addition, morphological parameters in protoscoleces of fertile cysts were measured to determine the relationship between the genotypes and morphometry. According to the obtained genotype and morphometry results, there were no statistically significant differences between the genotypes in terms of the number of hooks, total lengths of large and small hooks, blade lengths of large and small hooks, and widths of small hooks, although there was a statistically significant difference in large hook width (p > 0.05).

Biological and morphological consequences of dsRNA-induced suppression of tetraspanin mRNA in developmental stages of Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis, caused by the cestode Echinococcus granulosus, is a neglected tropical disease with remarkable morbidity in humans and a problem of worldwide economic importance in livestock industry. Understanding the molecular basis of the parasite growth and development is essential for the disease diagnosis, management and control. The tetraspanin (TSP) family of proteins are transmembrane proteins with a role in many physiological processes of eukaryotic organisms. TSPs present in the tegumental surface of platyhelminths play pivotal roles in host-parasite interaction. However, little is known about the role of TSPs in growth and development in the Platyhelminthes. To understand the role of TSP1 in the growth and development of E. granulosus we investigated the effect of EgTSP1-specific long dsRNA in different in vitro stages of the parasite. METHODS: Different stages of E. granulosus, protoscoleces and strobilated worms, were cultivated In vitro in di-phasic media. Using long dsRNA and two delivery methods, i.e. electroporation and electro-soaking, EgTSP1 silencing was performed with an EgTSP1-specific dsRNA. The TSP1 expression profile was assessed as well as the biological and ultrastructural properties of the parasites. RESULTS: After three days of dsRNA treatment, EgTSP1 expression was significantly reduced in both stages of E. granulosus as compared to irrelevant/unrelated dsRNA and untreated controls. Silencing expression of EgTSP1 in different stages of E. granulosus resulted in reduced viability and body contractions, inhibition of protoscoleces evagination and distinctive tegumental changes. Ultrastructural morphology of the strobilated worms treated with EgTSP1-specific dsRNA was indicative of the microtriches impairments and vacuolated tegument compared to the control helminths. CONCLUSIONS: Results of the present study suggest that EgTSP1 plays important structural roles in tegument configuration in E. granulosus. EgTSP1 is proved to be a potential target for the development of vaccines and RNAi-based drugs.

Cystic echinococcosis in Tunisia: analysis of hydatid cysts that have been surgically removed from patients.

Echinococcosis/hydatidosis caused by Echinococcus granulosus has a widespread distribution in the human population of Tunisia, particularly in the north-west and centre-west of the country. In a recent study, the morphological features, fertility and viability of hydatid cysts that had been excised from patients in Tunis were explored, and the E. granulosus strain or genotype involved in each case was identified from morphology of the protoscolex hooks and the results of molecular genotyping. The hepatic cysts investigated came from 41 patients [31 women and 10 men, with a mean (S.E.) age of 43.41 (14.25) years] who were treated for cystic echinococcosis, by surgery but rarely with chemotherapy, at the La Rabta Hospital in Tunis, in the 12 months ending in June 2008. Most (56%) of these patients originated from rural areas in endemic governorates. Of the 60 hepatic cysts that were studied, 38.3% were located in the right lobe of the liver and 35.0% each involved both hepatic lobes. Almost a third (31.7%) of the excised cysts were degenerating, with the rest considered viable and either multivesicular (38.3%) or univesicular (30.0%). Almost all (93.3%) of the cysts were categorized as fertile, with a mean protoscolex viability of 21.8%. Protoscolex viability was relatively high in the viable univesicular cysts with a visible cyst wall and in the multivesicular and multiseptate cysts with daughter cysts, and lowest in the cysts that appeared to be solid calcified masses. The observed variation in protoscolex viability with cyst type, in cysts excised from patients before any chemotherapy, supports the cyst classification recommended by the World Health Organization but could also be compatible with the imaging-based 'Gharbi' classification. The results of the molecular genotyping showed that all 23 cysts investigated (which came from 20 of the patients) were caused by E. granulosus of the G1 genotype (also known as the 'sheep' or 'sheep-dog' strain).

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.

DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts.

Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.

Stressor-induced changes to the protoscoleces of Echinococcus granulosus of Indian buffalo origin.

In the present study viable protoscoleces of Echinococcus granulosus were exposed to in vitro anthelmintic treatment to observe efficacy against Indian buffalo isolates. Evaluation criteria included morphological changes, viability scores and expression of peptides as a product of prestressed protoscoleces. Protoscolex changes included presence of bladder-like structure and morphological distortion. Two peptides of relative molecular weight (Mr) 40 and 70 kDa were visualized when proteins were separated by discontinuous gel electrophoresis. These two peptides seemed to be products of prestressed protoscoleces.

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.

Intraspecific variation of Echinococcus granulosus in livestock from Peru.

Echinococcus granulosus infection is a problem of public health in livestock-rearing regions of Latin America, like in Peru. This species is characterized by the morphometry of the large and small rostellar hooks in both metacestodes and adult worms. The aim of this work was to study biometrical characteristics of rostellar hooks in hydatid cysts (lung and liver) from some Peruvian provinces (Arequipa, Cuzco, Huancavelica, Junin and Puno). The biometrical analysis was obtained in a digital system for image analysis. The following parameters were studied: area, perimeter, length and width total of the handle, the blade and the distance between the blade and the guard of large and small hooks. For statistical analysis Student's t-test and Analysis of Variance (ANOVA) with Pos-test of Tukey were used, considering the values of p< or =0.05 as of statistic significance. All morphometric characters of large and small hooks were highly significant (p<0.000), when data were compared among the samples from the different provinces. Rostellar hooks in specimens from Huancavelica presented smallest measurements, whereas hooks in specimens from Junin were bigger in relation to the total and blade measurements. Generally, samples from Arequipa presented the biggest large hooks in relation to the handle parameters. The rostellar hooks of cattle and sheep hosts varied depending on the geographic localization, host and organ. Hooks recovered from the lungs were larger than those collected in the liver. It was concluded that morphometry using computer image analysis allows the characterization of E. granulosus. The rostellar hooks presented morphometric polymorphism.

[Identification of the causative agent of echinococcosis in the population of the South Urals].

The topicality of the problem associated with echinococcosis granulosus in the South Urals is determined by its wide spread and a considerable economic damage made to this region by this invasion. The study was undertaken to reveal the intraspecific affiliation of Echinococcus granulosus that induces hydatid disease in the population of the South Urals. Samples for studies were taken from the fertile larval cysts obtained during intraoperative intervention in patients with hydatid disease. As morphological criteria for differentiation, the authors examined the proboscis uncuses of protoscolexes. For E. granulosus genomic typing, polymerase chain reaction (PCR) of DNA synthesis was used, as described by Gasser (1998). As a DNA marker, the authors used a fragment of the mitochondrial gene encoding for the first subunit of cytochome-C-oxidase. The DNA fragments obtained by PCR from 9 isolated underwent the direct enzyme dideoxy-sequencing test (Senger, 1977). As a result, the causative agent of echinococcosis granulosis was first identified in the patient of the South Urals. In children and adults, the clinical form of the disease is caused by E. granulosus with the genotype G - common, that of domestic sheep. Comparative analysis of molecular data revealed the presence of genotype G1 variations circulating in the South Urals homologous to the sequences recorded in the GenBank under numbers U50464 and DQ109036.

Estudio morfocuantitativo de protoescólices y otras estructuras fundamentales de Echinococcus granulosus en hospederos humanos / Morphoquantitative study of protoscolices and other fundamental structures of Echinococcus granulosus in human hosts

La equinococosis quística (EQ) a pesar de ser una enfermedad endémica en diversos lugares del planeta, presenta pocos estudios morfológicos y cuantitativos de las estructuras fundamentales del Echinococcus granulosus en humanos, en especial de los protoescólices. El objetivo de este estudio fue analizar morfocuantitativamente protoescólices y otras estructuras fundamentales de E. granulosus obtenidos de hospederos humanos. Estudio de corte transversal. Se estudiaron 8 especímenes de EQ hepática humana, aplicando un muestreo no probabilístico por conveniencia. Se evaluó las capas quísticas, el líquido y la arenilla hidatídica. Las capas fueron fijadas en formaldehido tamponado a 10 % y procesadas para su inclusión en paraplast. Se realizaron cortes de 5 µm de grosor y fueron teñidas con H-E para su análisis con microscopía óptica. El líquido y arenilla fueron centrifugados y al sedimento obtenido se le realizó análisis directo para determinar las medidas morfométricas de los protoescólices y de los ganchos grandes y pequeños. Se utilizó estadística descriptiva. De los 8 quistes estudiados, 6 eran quistes univesicular, uno multivesicular y un quiste abscedado, cuyas capas laminada y germinativa se encontraban bien definidas. Las vesículas prolígeras presentaban forma redondeada con protoescólices en su interior. Los protoescólices invaginados presentaron un largo y ancho promedio de 140,8 ± 34,3 µm y 106,2 ± 29,5 µm, respectivamente; y los desarrollados un largo de 237,2 ± 53,0 µm y ancho de 128,7 ± 32,0 µm. Los ganchos rostelares presentaron contornos suaves distribuidos en dos filas regulares. El promedio del largo total de los ganchos grandes y pequeños fue 20,1 ± 2,7 µm; el promedio del ancho total fue 7,4 ± 1,2 µm. En conclusión, las características morfocuantitativas de los ganchos de E. granulosus en humanos, son distintos a otras especies hospederas intermediarias y de otros Echinococcus spp. Es posible que el abandono del estado de resistencia de los protoescólices invaginado hasta su desarrollo genere implicancias epidemiológicas de interés.

SUMMARY: Although cystic echinococcosis (CE) is an endemic disease in several parts of the world, few morphological and quantitative studies of the fundamental structures of Echinococcus granulosus in humans, especially protoscolices. The aim of this study was to perform a morphoquantitative analysis of protoescolex and other fundamental structures of E. granulosus from human hosts. Cross- sectional study. Eight human hepatic EQ specimens were studied, applying non-probabilistic convenience sampling. Cystic layers, fluid and hydatid grit were evaluated. The layers were fixed in 10% buffered formaldehyde and processed for embedding in paraplast. Slices of 5 µm thickness were made and stained with H-E for light microscopic analysis. The liquid and grit were centrifuged and the sediment obtained was analyzed directly to determine the morphometric measurements of the protoscolices and the large and small hooks. Descriptive statistics were used. Of the 8 cysts studied, 6 were univesicular cysts, one multivesicular and one abscessed cyst, whose lamellar and germinative layers were well defined. The proligerous vesicles had a rounded shape with protoscolices inside. The invaginated protoscolices had an average length and width of 140.8 ± 34.3 µm and 106.2 ± 29.5 µm, respectively; and the developed ones had a length of 237.2 ± 53.0 µm and width of 128.7 ± 32.0 µm. The rostellar hooks presented smooth contours distributed in two regular rows. The average total length of the large and small hooks was 20.1 ± 2.7 µm; the average total width was 7.4 ± 1.2 µm. In conclusion, the morphoquantitative characteristics of E. granulosus hooks in humans are distinct from other intermediate host species and from other Echinococcus spp. It is possible that the abandonment of the resistance state of the invaginated protoscolices until their development generates epidemiological implications of interest.

Characterization of Echinococcus granulosus isolates from human, sheep and camel in Iran.

In the present study, Echinoccocus granulosus isolates collected from human, sheep and camel samples in Iran were characterized based on rostellar hook morphology of protoscoleces as well as PCR-RFLP. Morphological study on human and animal isolates showed the presence of two distinct strains of the parasite, one in sheep and the other one in camels. In this regard, rostellar hook of sheep isolates were significantly different from those of camel origin, meanwhile human isolates were found to be similar to those isolated from sheep. Molecular analysis of the ITS1 region of rDNA derived from human, sheep and camel isolates were in agreement with the morphological findings. Based on the PCR-RFLP method, the sheep and human isolates appeared to pertain to the same genotype and the camel isolates were appeared to pertain to a different genotype.

Morphological and molecular characteristics of Echinococcus multilocularis and Echinococcus granulosus mixed infection in a dog from Xinjiang, China.

The Xinjiang plateau of western China has been shown to have a high prevalence for human cystic echinococcosis (CE) caused by Echinococcus granulosus, and human alveolar echinococcosis (AE) caused by Echinococcus multilocularis. The domestic dog is suspected to be the primary definitive host for the transmission of both E. granulosus and E. multilocularis to humans in this locality. Seventeen of 30 stray dogs from Hejing County of Xinjiang were found positive for E. granulosus post mortem, and one double infection was suspected. Worm samples were collected, dyed by carmine, and observed microscopically. Carmine staining examination clearly revealed the differences in number of proglottids and appearance of uterine branches and lateral genital pore for those two species of Echinococcus. Furthermore, gene target DNA fragments were amplified for formal identification of the two parasite species, based on 12s rRNA mitochondrial gene. The PCR products were purified and sequenced. Compared with NCBI GenBank, the DNA sequences demonstrated 100% identity with E. granulosus (sheep strain, G1 genotype) and E. multilocularis.

[Mixed infection of Echinococcus granulosus and Echinococcus multilocularis in dog].

OBJECTIVE: To identify mixed infection of Echinococcus granulosus and E. multilocularis in a dog from Xinjiang. METHODS: Thirty dogs from the pasture area were dissected and over 10,000 Echinococcus adult worms were found from one dog. Morphological observation revealed possible mixed infection of the two Echinococcus species. Further identification was made by amplification of the target gene DNA fragment (mitochondrial 12S rRNA gene). RESULTS: The adult worms of E. granulosus showed a relatively longer and larger gravid proglottid, its genital pore situated near or below the middle-side of the segment. The uterus was in a sacculate shape with irregular branches and approximately over 200 - 800 eggs in it. Morphology of the adult worms of E. multilocularis was similar to E. granulosus, slightly smaller, consisting of 4 to 5 proglottids. The uterus was not sacculate and with no branch. Its lateral genital pore often situated in the anterior part of the segment. Sequence analysis of mitochondrial 12S rRNA gene showed that amplification with the Eg1f/r primers shared complete identity with E. granulosus G1 genotype (GenBank accession no. AY462129), while that witht the EmH15/17 primers shared complete identity with E. multilocularis (GenBank accession no. AB031351). The presence of both E. granulosus and E. multilocularis was confirmed by microscopy and gene identification. CONCLUSION: Mixed infection of the two species of Echinococcus has been confirmed in the dog by morphological observation and PCR technique.

The nervous and prenervous roles of serotonin in Echinococcus spp.

Serotonin (5-hydroxytryptamine, 5-HT) is an important neuroactive and morphogenetic molecule in several metazoan phyla, including flatworms. Serotoninergic nervous system studies are incomplete and 5-HT function/s are unknown in Echinococcus spp., the flatworm parasites that cause hydatid disease. In the present work, we searched for genes of the serotoninergic pathway and performed immunocytochemical and functional analyses of 5-HT in Echinococcus spp. Bioinformatic analysis using the recently available Echinococcus multilocularis and Echinococcus granulosus genomes suggests the presence of genes encoding enzymes, receptors and transporters participating in 5-HT synthesis, sensing and transport in these parasites. However, some components of the pathway could not be identified, suggesting loss or divergence of parasite homologous genes. The serotoninergic neuroanatomy study performed by confocal scanning laser microscopy on different E. granulosus stages showed an increasing level of complexity when the protoscolex develops towards the adult stage and a progressive diminution when the parasite develops towards the metacestode stage. The role of 5-HT as a neurotransmitter in E. granulosus was evaluated by determining the effect of this substance on protoscolex motility. The addition of 5-HT to protoscoleces induced a significant increase in motility for short time periods. Preincubation with 100 µM citalopram, a known 5-HT transporter inhibitor, abolished the 5-HT-induced increase in motility, indicating that the effect could be mediated by a 5-HT transporter. Incubation of protoscoleces with 5-HT for time periods of several days induced a progressive differentiation towards the metacestode stage. The results indicate that 5-HT could have nervous and prenervous roles during Echinococcus spp. development. Taking into account the important roles of 5-HT in parasite biology and the divergence of 5-HT pathway genes with respect to human counterparts, the serotoninergic system could be considered as an amenable drug target against hydatid disease.