Echinococcus granulosus: Protoscolicidal effect of high intensity focused ultrasound

High intensity focused ultrasound (HIFU) is a new non-invasive technique which can cause cell death and tissue necrosis by focusing high-energy ultrasonic waves on a single location. The aim of our work is to investigate the damaging effect of HIFU on Echinococcus granulosus protoscolices, as well as its inhibitory effect on growth of hydatid cysts derived from protoscolices. The damaging effect of HIFU on protoscolices was investigated by following parasite mortality after irradiation, while the inhibitory effect was investigated by infection experiments in vivo. The results demonstrated that HIFU was able to damage protoscolices and the protoscolicidal effect was dose-dependent and showed late-onset. The growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro, and the mean weight of hydatid cysts resulting from such protoscolices in the experimental group was less than that in controls. Evidences including the protoscolicidal effect, fragmentized protoscolices and low post exposure temperatures, suggest that cavitation may contribute to the protoscolicidal effect of HIFU. In addition, the structure of the germinal membrane in cysts developing from the irradiated protoscolices was not as normal or intact as that from non-irradiated ones, and morphological changes related to degeneration were observed, suggesting that HIFU could prevent protoscolices from developing normal germinal membrane and consequently stop the proliferation of secondary hydatid cysts. HIFU demonstrated damaging effect on protoscolices, inhibited the growth of protoscolices in vitro and in vivo, and could be a possible therapeutic option for cystic echinococcosis.     

Evidence that the Echinococcus granulosus G6 genotype has an affinity for the brain in humans

The present study investigates the molecular characteristics of cerebral Echinococcus cysts. A total of 10 specimens of cerebral Echinococcus cysts, including six formalin-fixed paraffin blocks and four intact cerebral cysts, were used for this study. The target DNA was successfully amplified from eight samples and sequenced. BLAST analysis indicated that sequenced isolates belong to the Echinococcus granulosus (G6) genotype. All of the eight sampled brain cysts belonged to the G6 genotype, while all of the eight liver cysts belonged to G1. This is a strong indication that G6 has a higher affinity for the human brain than G1.     

Modelling the age variation of larval protoscoleces of Echinococcus granulosus in sheep

In autumn 2006, a study of the age-dynamics of Echinococcus granulosus cyst abundance was undertaken from an abattoir study of 1081 sheep slaughtered in Naryn Province in central Kyrgyzstan, an area endemic for echinococcosis. The results demonstrated approximately 64% of sheep were infected with the prevalence increasing markedly with age. The mean abundance was 3.8 cysts per sheep. From established models, an infection pressure of 1.33 cysts per year was estimated. In addition all cysts were recovered from infected sheep and the numbers of protoscoleces was evaluated in each cyst. A new model was developed that examined the variation in numbers of protoscoleces per infected sheep with age. This demonstrated that young sheep aged 1-2 years had very few protoscoleces, but there was a massive increase as the sheep aged. The best-fitting model assumed that the number of protoscoleces in a sheep was proportional to the volume of the cysts. In this model, the radius of the individual cyst increased linearly with the age of the cyst and hence the volume increased with the cube of the cyst age. This combined with the linear increase in numbers of cysts with age resulted in a massive accumulation of protoscoleces with the age of sheep. When the model was parameterised it demonstrated that 80% of protoscoleces were present in sheep aged 4 years and older and this represented just 28% sheep slaughtered. An average sheep at 6 or more years of age has an abundance of over 9700 protoscoleces, whilst in a young sheep of 1 year of age an average of just 16 protoscoleces could be found. The average for the sampled population across all ages was 1562 protoscoleces per sheep. The maximum number of protoscoleces in a single cyst was just 482 for sheep aged 1 year rising to 92,000 for sheep aged 6 years or older. The mean volume of cysts containing protoscoleces increased from approximately 0.7 ml at 1 year of age to 8.8 ml at 6 years of age. Cysts containing protoscoleces ranged from a diameter of 0.5-8 cm with a volume of fluid ranging from 0.2 to 50 ml. It is hypothesised that removal of old sheep through a culling programme could substantially improve the control of cystic echinococcosis.     

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.     

Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran.Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.     

First report of a mixed infection due to Acanthamoeba genotype T3 and Vahlkampfia in a cosmetic soft contact lens wearer in Iran

Acanthamoeba keratitis cases have emerged in the recent years in Iran. In this case, an amoebic keratitis due to a mixed infection with Acanthamoeba and Vahlkampfia species is reported. Corneal scrapes, contact lenses and contact lens cases obtained from the patient were analysed and were positive for cysts of Acanthamoeba and Vahlkampfia genera. Genus-specific PCR was carried out for both genera, confirming the microscopic observations. To our knowledge, this is the first reported case of a possible mixed amoebic infection due to Acanthamoeba and Vahlkampfia and raises awareness within contact lens wearers in Iran.     

Echinococcus granulosus sensu lato genotypes infecting humans – review of current knowledge

Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1–10 and the ‘lion strain’), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1–3 (=E. granulosus sensu stricto) and G6–10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1–3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6–7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by the G5, G8 and G10 genotypes. No cases of human infection with G4 have been described. Biological differences between the species and genotypes have potential to affect the transmission dynamics of the parasite, requiring modification of methods used in disease control initiatives. Recent investigations have revealed that the protective vaccine antigen (EG95), developed for the G1 genotype, is immunologically different in the G6 genotype. Further research will be required to determine whether the current EG95 vaccine would be effective against the G6 or G7 genotypes, or whether it will be necessary, and possible, to develop genotype-specific vaccines.     

Echinococcus multilocularis: Single hepatic lesion experimentally established without metastasis in rats

We herein describe the establishment of single hepatic lesions of Echinococcus multilocularis in rats. A 3 mm incision was made on the liver with a surgical knife, and one small round vesicle of E. multilocularis (between 1 × 1 mm and <2 × 2 mm in diameter) was transplanted into the incision and covered with absorbable hemostat gauze. The presence and growth of the transplanted vesicle was monitored for 12 weeks using magnetic resonance imaging (MRI). Hepatic lesions, the metacestode of this parasite were confirmed in 12 of 17 infected rats (70.6%) by MRI and macroscopic examinations. The average size of the metacestodes with brood capsules at 12 weeks after the experimental transplantation of a single vesicle was 6.1 ± 2.5 mm × 4.4 ± 1.5 mm. The smallest size of the metacestodes detected by MRI was approximately 3 × 3 mm. This new approach of establishing single hepatic metacestodes of E. multilocularis in experimental animals is expected to be useful for analyzing the immune-pathological mechanisms of hepatic AE.     

Echinococcus granulosus: In vitro effectiveness of warm water on protoscolices

Hydatid disease is one of the most important helminthic diseases worldwide. Hydatid cysts may be found anywhere in the body. The most effective treatment of hydatid cyst is surgical operation. Spillage of live protoscolices during the operation is the major cause of recurrence. Instillation of scolicidal agent into hydatid cyst is the most commonly employed measure to prevent this complication. To date, many scolicidal agents have been used for inactivation of the hydatid cyst content, however, most common scolicidal agents may cause unacceptable side-effects, limiting their use. In this study the scolicidal effect of warm water (45, 50, 55, and 60 °C) at different exposure times (1, 2, 3, 4, 5, 6, 8, 10, 12, and 15 min) is investigated. Protoscolices were collected aseptically from sheep livers containing hydatid cyst. Viability of protoscolices was determined by 0.1% eosin staining. Even though the highest scolicidal activity of warm water at 45 °C was 40.4% at the end of 15 min, the best scolicidal effect (100%) of warm water at 50, 55, and 60 °C was obtained after 5, 2, and 1 min, respectively. The results of this in vitro study showed that warm water at 50–60 °C can be regarded as an effective scolicidal agent. Warm water is commonly available, easily prepared, and inexpensive. In vivo scolicidal activity of warm water and also the possible side effects need further investigation.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.     

Fruit anatomy of the genus Pimpinella L. (Apiaceae) in Iran

The genus Pimpinella L., with about 170–180 species in the world, is one of the largest genera of the family Apiaceae (Umbelliferae). Based on the Flora Iranica treatment, this genus has 25 species in the Iranian plateau, including 19 species in Iran, and six of those (P. tragioides, P. deverroides, P. pastinacifolia, P. anisactis, P. khorasanica and P. khayyamii) are endemic.     

Apparent dominance of the G1–G3 genetic cluster of Echinococcus granulosus strains in the central inland region of Portugal

Infection by the larval stage of the cestode Echinococcus granulosus causes a disease known as cystic echinococcosis or hydatidosis, which is one of the most widespread zoonotic infections of veterinary and medical importance. Numerous studies have shown that E. granulosus exists as a complex of strains differing in a wide variety of criteria. Ten distinct genotypes (G1–G10) have been identified with a potential impact on the pathology, epidemiology and the effect of the measures implemented for the control of hydatidosis. Our main objective was to carry out a preliminary analysis of the genotypes of E. granulosus circulating in the central inland region of Portugal.Parasite samples (hydatid cysts, n = 27) were isolated from the liver and lung of sheep and cattle. The DNA extracted from protoscoleces isolated from the fertile cysts served as a template for the PCR amplification of the part of the mitochondrial cytochrome c oxidase subunit 1 (cox1), ATP synthase F0 subunit 6 (atp6) as well as the large (rrnL/16 S) and small (rrnS/12 S) ribosomal RNA genes. Similarity searches with homologous sequences in the databanks indicated a very high similarity with references assigned to the G1, G3 and/or G1–G3 complex of Echinococcus strains. Phylogenetic analysis (Bayesian approach) supported these observations, and confirmed the assignment of all the analyzed sequences to the G1–G3 genetic cluster.     

The origin of a new focus of infection with Echinococcus granulosus in tasmania

Hydatid cysts were discovered in cattle on King Island, north of Tasmania, where Echinococcus granulosus was thought to have been eradicated. Using enzyme electrophoresis, isolates from King Island were compared genetically with isolates from Tasmania and the mainland of Australia. The genetic distinctness of the King Island isolates make it unlikely that they originated from a recent introduction from either Tasmania or mainland Australia. Alternative possibilities, that the infection resulted from a recent introduction from another source or from previously undetected persistence of E. granulosus on King Island, could not be distinguished from available data.     

Echinococcus granulosus cysts: Early development in vitro in the presence of serum from infected sheep

When cultured in vitro in the presence of serum from a number of sheep infected with Echinococcus granulosus cysts, varying proportions of oncospheres died within 24 h. Of the survivors, some died during reorganization into cysts; others were able to develop normally but showed evidence of precipitates in the outer layers of the cyst. The lethal effects were removed by heating the serum to 56°C for 30 min and could be restored by the addition of freshly-collected normal sheep serum. In the presence of serum from sheep immunized against E. granulosus, most oncospheres were dead within 24 h, and few or none of the survivors were able to reorganize into cysts.     

Spatial heterogeneity and temporal variations in Echinococcus multilocularis infections in wild hosts in a North American urban setting

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, has the potential to circulate in urban areas where wild host populations and humans coexist. The spatial and temporal distribution of infection in wild hosts locally affects the risk of transmission to humans. We investigated the spatial and temporal patterns of E. multilocularis infection in coyotes and rodent intermediate hosts within the city of Calgary, Canada, and the association between spatial variations in coyote infection and the relative composition of small mammal assemblages. Infection by E. multilocularis was examined in small mammals and coyote faeces collected monthly in five city parks from June 2012 to June 2013. Coyote faeces were analysed using a ZnCl₂ centrifugation and sedimentation protocol. Infection in intermediate hosts was assessed through lethal trapping and post-mortem analysis. Parasite eggs and metacestodes were morphologically identified and molecularly confirmed through species-specific PCR assays. Of 982 small mammals captured, infection was detected in 2/305 (0.66%) deer mice (Peromyscus maniculatus), 2/267 (0.75%) meadow voles (Microtus pennsylvanicus), and 1/71 (1.41%) southern red backed voles (Myodes gapperi). Overall faecal prevalence in coyotes was 21.42% (n = 385) and varied across sites, ranging from 5.34% to 61.48%. Differences in coyote faecal prevalence across sites were consistent with local variations in the relative abundance of intermediate hosts within the small mammal assemblages. Infections peaked in intermediate hosts during autumn (0.68%) and winter (3.33%), and in coyotes during spring (43.47%). Peaks of infections in coyote faeces up to 83.8% in autumn were detected in a hyper-endemic area. To the best of our knowledge, our findings represent the first evidence of a sylvatic life-cycle of E. multilocularis in a North American urban setting, and provide new insights into the complexity of the parasite transmission ecology.     

Multi-locus microsatellite analysis supports the hypothesis of an autochthonous focus of Echinococcus multilocularis in northern Italy

Echinococcus multilocularis is characterised by a wide geographical distribution, encompassing three continents (North America, Asia and Europe) yet very low genetic variability is documented. Recently, this parasite has been detected in red foxes (Vulpes vulpes) circulating in an Alpine region of Italy, close to Austria. This finding raised the question as to whether an autochthonous cycle exists in Italy or whether the infected foxes originated from the neighbouring regions of Austria. Studies have shown that multi-locus microsatellite analysis can identify genomic regions carrying mutations that result in a local adaptation. We used a tandem repeated multi-locus microsatellite (EmsB) to evaluate the genetic differences amongst adult worms of E. multilocularis collected in Italy, worms from neighbouring Austria and from other European and extra-European countries. Fluorescent PCR was performed on a panel of E. multilocularis samples to assess intra-specific polymorphism. The analysis revealed four closed genotypes for Italian samples of E. multilocularis which were unique compared with the other 25 genotypes from Europe and the five genotypes from Alaska. An analysis in the Alpine watershed, comparing Italian adult worms with those from neighbouring areas in Austria, showed a unique cluster for Italian samples. This result supports the hypothesis of the presence of an autochthonous cycle of E. multilocularis in Italy. EmsB can be useful for ‘tracking’ the source of infection of this zoonotic parasite and developing appropriate measures for preventing or reducing the risk of human alveolar echinococcosis.     

Limit of detection of sedimentation and counting technique (SCT) for Echinococcus multilocularis diagnosis, estimated under experimental conditions

The aim of this study was to estimate the limit of detection of sedimentation and counting techniques (SCT) in Echinococcus multilocularis diagnosis. Samples of small intestines, experimentally enriched with known numbers of E. multilocularis tapeworms, were used. Forty intestinal samples containing 2, 5, 10, and 30 E. multilocularis tapeworms (10 samples for each level) were prepared and examined according to SCT. E. multilocularis was detected in 30%, 40%, 60%, and 100% in samples enriched with 2, 5, 10, and 30 tapeworms, respectively. The limit of detection was estimated at 10 E. multilocularis tapeworms per sample of intestine (for 60% probability of obtaining positive results). There was a wide dispersion of counting results; these were observed in samples containing the same numbers of tapeworms, which indicates the low repeatability of the method. The limitations of SCT determined in this experiment should be considered when analysing the prevalence of E. multilocularis in carnivores.