Assessment of the epidemiological status of Echinococcus multilocularis in foxes in France using ELISA coprotests on fox faeces collected in the field

The aim of this study was to estimate the relevance of Echinococcus multilocularis coproantigen detection in fox faeces collected in the field to identify different levels of endemicity for Echinococcus multilocularis on a large scale (n×10 km²). Six study sites were selected in a high endemicity area and two study sites in a low endemicity area in eastern France on the basis of landscape composition. Sampling was undertaken in the winters of 1996–97, 1997–98 and 1998–99. At each site, (i) necropsy and intestine examination was undertaken on a sample of shot foxes (total number of foxes, 222), and (ii) fox faeces were collected in the field along road verges, and scored for degradation status (total number of faeces, 625). Fox faeces were also sampled in a control area (n=30) in western France in the summer of 1998. Intestines were examined according to the sedimentation method. Echinococcus multilocularis coproantigens were detected by using two ELISA tests: EM-ELISA and EmA9-ELISA. The necropsy prevalence in high and low endemicity areas was 63.3% and 19.4%, respectively, and the distribution of adult worms in the fox population was highly overdispersed (75.5% of the total biomass was harboured by 11.6% of foxes). Using the two ELISA tests, there was no difference in the detection of E. multilocularis coproantigens in field faeces, regardless of the degradation status. The medians of EM- and EmA9-ELISA OD values of field faeces in high endemicity area were significantly higher than in low endemicity area (P<0.001 for both ELISA). The distribution of EM-ELISA OD values in low endemicity area was significantly higher (P=0.002) than in the control area. Moreover, for the two ELISA, the observed ELISA OD value distributions in high endemicity area, low endemicity area and control area seemed representative of the distribution of adult worms in fox populations. These results indicate that E. multilocularis coproantigen detection in field faeces could serve for large-scale surveillance, as an alternative to necropsy.     

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: 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.     

Purine metabolism in Echinococcus multilocularis

The activities of the enzymes in Echinococcus multilocularis metacestodes involved in purine salvage were studied by HPLC. As in most parasites, this cestode relies entirely on salvage of preformed bases and nucleosides for its purine requirement. Therefore, these enzymes may be targets for drugs in the chemotherapeutic treatment of diseases caused by this parasite. The animals used in this study were gerbils (Meriones unguiculatus). Enzyme activities from sera and hepatic tissue in control and infected animals were similar with the exception of adenine phosphoribosyltransferase which showed an activity 4-fold greater in the serum from control than in serum from infected animals. In the parasite, adenine and hypoxanthine-guanine phosphoribosyltransferases and adenosine deaminase had the highest activities. Therefore, in E. multilocularis metacestodes, this pathway seems to be important for the parasite’s metabolism.     

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.     

Arvicola terrestris an intermediate host of Echinococcus multilocularis in France: Epidemiological consequences

Houin R., Deniau M., Liance M. and Puel F. 1981. Arvicola terrestris, an intermediate host of Echinococcus multilocularis in France: epidemiological consequences. International Journal for Parasitology12: 593–600. Hydatid cysts characteristic of E. multilocularis were found in 5 out of 2010 A. terristris trapped in France in 1980. A comparison of the distribution of human cases and rodents led to the conclusion that A. terrestris is probably the reservoir of the parasite. Another argument in favour of the epidemiological importance of this vole is that the parasite grows slowly in this host which has a relatively long life. Moreover, no other rodents were found in France naturally infected by larval E. multilocularis. As populations of A. terrestris undergo important cyclical fluctuations, high transmission periods must occur. During the periods of peak population, prophylactic measures should be taken to protect the human population.     

Phylogeographic patterns in Leccinum sect. Scabra and the status of the arctic-alpine species L. rotundifoliae

We investigated inter- and intraspecific phylogenetic relationships in the ectomycorrhizal fungal genus Leccinum section Scabra. Species of this section are exclusively associated with Betula and occur throughout the Northern Hemisphere. We compared the phylogenetic relationships of arctic, alpine, boreal and temperate accessions of section Scabra based on DNA sequences of the single-copy nuclear gene Gapdh and the multiple-copy nuclear region 5.8S-ITS2. Exclusively arctic lineages were not detected in species that occur both in arctic-alpine or boreal regions, except in L. rotundifoliae that was restricted to cold climates. L. scabrum and L. holopus showed an intercontinental phylogeographic pattern, and L. variicolor showed a pattern unrelated to geographical distribution. Molecular clock estimates indicated that L. rotundifoliae is as old as other species in section Scabra. Individual gene trees suggest that interspecific hybridisation occurred several times in the evolution of section Scabra.     

Echinococcus multilocularis in the mouse: The in vitro protoscolicidal activity of peritoneal macrophages

Baron R. W. and Tanner C. E. 1977. Echinococcus multilocularis in the mouse: the in vitro protoscolicidal activity of peritoneal macrophages. International Journal for Parasitology7: 489–495. The larvae of Echinococcus multilocularis are susceptible to the protoscolicidal activity of infected A/J mouse peritoneal cells. It is shown that the effector cell in this response is an activated macrophage. Preincubation of protoscolices in ‘immune’ serum increases their susceptibility to the protoscolicidal activity of infected mouse peritoneal cells. Macrophages activated nonspecifically by BCG or Taenia crassiceps infections also exhibit protoscolicidal activity in vitro. The identity of the effector cell was confirmed by scanning electron microscopy. It is shown that ‘immune’ macrophages adhere to and form close cellular contacts with the protoscolex surface. It is concluded that resistance to hydatid infections is mediated by activated macrophages.     

Segmental sedimentation and counting technique (SSCT): an adaptable method for qualitative diagnosis of Echinococcus multilocularis in fox intestines

A modified Segmental Sedimentation and Counting Technique (SSCT) to examine the presence of Echinococcus multilocularis helminths in segments of fox (Vulpes vulpes) intestine is described and compared to the “gold standard”, SCT. Out of the 358 intestines collected, 117 were E. multilocularis positive. Using SSCT methods we compare the sensitivity of individual or pairs of segments to establish a tradeoff between saving time and the reliability of the diagnosis, especially in areas with low infection intensities. The results show that the analysis of segment S4 associated with segment S1 or S2 give 98.3% sensitivity, with specificity close to 100%. Based on our results and the time saved, we recommend using SSCT for routine examination of fox intestines for large epidemiological studies, particularly where the endemic prevalence of E. multilocularis is low or unknown.     

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.     

Ecosystem implications of genetic variation in water-use of a dominant riparian tree

Genetic variation in dominant species can affect plant and ecosystem functions in natural systems through multiple pathways. Our study focuses on how genetic variation in a dominant riparian tree (Populus fremontii, P. angustifolia and their natural F₁ and backcross hybrids) affects whole-tree water use, and its potential ecosystem implications. Three major patterns were found. First, in a 12-year-old common garden with trees of known genetic makeup, hybrids had elevated daily integrated leaf-specific transpiration (E_tl ; P=0.013) and average canopy conductance (G_c ; P=0.037), with both E_tl and G_c ~30% higher in hybrid cross types than parental types. Second, ¹³C values of leaves from these same trees were significantly more negative in hybrids (P=0.004), and backcross hybrids had significantly more negative values than all other F₁ hybrid and parental types (P <0.001). Third, in the wild, a similar pattern was found in leaf ¹³C values where both hybrid cross types had the lowest values (P <0.001) and backcross hybrids had lower ¹³C values than any other tree type (P <0.001). Our findings have two important implications: (1) the existence of a consistent genetic difference in whole-tree physiology suggests that whole-tree gas and water exchange could be another pathway through which genes could affect ecosystems; and (2) such studies are important because they seek to quantify the genetic variation that exists in basic physiological processes—such knowledge could ultimately place ecosystem studies within a genetic framework.     

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κ_m values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κ_i value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κ_i value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.     

横断山区干旱河谷川滇蔷薇种子休眠与萌发的地理空间差异

通过测定横断山区干旱河谷18个川滇蔷薇种群新采集种子以及低温层积8周种子的发芽率和发芽速度,分析发芽率和发芽速度与种子特征以及环境因子之间的关系,阐明川滇蔷薇种子休眠与萌发的地理空间差异及其影响因素。结果表明,18个川滇蔷薇种群种子具有不同程度的休眠,新采集种子发芽率变化幅度大,为15.8%±5.0%至82.7%±2.3%,发芽速度指数范围：2.3%±0.2%至5.3%±0.5%。不同种群种子发芽率和发芽速度差异显著。新采集种子的发芽率在流域间存在显著差异,表现为金沙江流域 > 雅砻江流域 > 大渡河流域和岷江流域。新采集种子的发芽率和发芽速度随着采集地点海拔的升高而显著增加,即种子休眠程度随着海拔的升高呈现降低趋势。低温层积8周显著提高了种子发芽率和发芽速度,但减弱了种子发芽率和发芽速度在流域以及海拔间的差异。偏相关分析表明：瘦果皮厚度与新采集种子萌发速度成显著负相关关系;环境因子中年蒸发量与发芽率以及发芽速度之间关系最为密切,成极显著正相关关系;其次为年降水量,与发芽率和发芽速度之间具有显著的负相关关系。综合分析表明,川滇蔷薇种子休眠与萌发在横断山区干旱河谷存在较强地理空间差异。环境因子中年蒸发量和年降水量以及植物自身特征瘦果皮厚度是引起种子休眠与萌发地理空间差异的主要因素。     

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.     

Echinococcus multilocularis: Two-dimensional Western blotting method for the identification and expression analysis of immunogenic proteins in infected dogs

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine.     

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.     

Echinococcus multilocularis phosphoglucose isomerase (EmPGI): A glycolytic enzyme involved in metacestode growth and parasite-host cell interactions

In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.