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.     

A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis

Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes.     

Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry

Echinococcus multilocularis is an important parasite that causes human alveolar echinococcosis. Identification and characterization of the proteins encoded by E. multilocularis metacestode might help to understand the complexity of the parasites and their interactions with the host, and to identify new candidates for immunodiagnosis and vaccine development. Here we present a proteomic analysis of E. multilocularis protoscolex (PSC) proteins. The proteins were resolved by 2-DE (pH range 3.5-10), followed by MALDI-TOF MS analysis. Fourteen known Echinococcus proteins were identified, including cytoskeletal proteins, heat shock proteins, metabolic enzymes, 14-3-3 protein, antigen P-29 and calreticulin. To construct a systematic reference map of the immunogenic proteins from E. multilocularis PSC, immunoblot analysis of PSC 2-DE maps was performed. Over 50 proteins spots were detected on immunoblots as antigens and 15 of them were defined. The results showed that cytoskeletal proteins and heat shock proteins were immunodominant antigens in alveolar echinococcosis.     

Fine structure and freeze-etch study of the protoscolex tegument of Echinococcus multilocularis (cestoda)

Freeze-etch replicas of the protoscolex tegument of Echinococcus multilocularis were examined and compared with conventional thin sections by TEM. The microtopography of the protoscolex tegument was also examined by SEM. The protoscolex consisted of morphologically-distinct, apical and basal tegumentary regions, the latter of which lacked microtriches. The hook area of the apical region contained long, slender, filamentous microtriches that obscured the hook arrangement. These microtriches were structurally different from those found on the suckers and rostellum of the protoscolex. Freeze-etch replicas of the tegumental membrane of the sucker and rostellar microtriches showed that the protoplasmic (P) and exoplasmic (E) faces of the microthrix base and tip contained numerous intramembranous particles (IMP). The densities of the IMP on both the P and E faces of the microthrix tip were approximately twice the number of the larger diameter IMP found on the P and E faces of the microthrix base. No freeze-etch replicas of the microtriches from the hook area were obtained. The basal tegumentary region of the protoscolex consisted of irregularly-distributed, knoblike processes that were variable in size and shape, and contained an electron-dense cap. The IMP on the P face of the knoblike processes measured approximately the same diameter as those on the P face of the microthrix base. However, their density was about half that of the latter. The density of IMP on the E face of the knoblike processes could not be determined from the freeze-etch replicas.     

Echinococcus multilocularis in the cotton rat: the in vitro protoscolicidal activity of peritoneal cells.

Protoscolices of Echinococcus multilocularis were incubated in vitro with peritoneal cells and with sera from normal and infected cotton rats. The cells from rats bearing massive subcutaneous and intraperitoneal hydatid cysts rapidly killed protoscolices; normal cells and cells from lightly infected animals did not. Only sera from rats bearing large, subcutaneous cysts displayed significant protoscolicidal activity. These data suggest that the phenomenon whereby large, established cysts of E. multilocularis effectively suppress the establishment, growth, and metastasis of distant foci of infection has an immunological component.     

The lipids and phospholipids of hydatid protoscolices of Echinococcus granulosus (cestoda)

Frayha G. J., Bahr G. M. and Haddad R. 1980. The lipids and phospholipids of hydatid protoscolices of Echinococcus granulosus (Cestoda). International Journal for Parasitology10: 213–216. The protoscolices of Echinococcus granulosus were isolated from hydatid cysts. The phospho- and neutral lipids of the protoscolices were determined. The seven major classes of lipids namely, phospholipids, fatty acids, mono-, di- and triglycerides, cholesterol and cholesterol esters were identified and quantitatively determined. The phospholipids isolated were cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. Lecithin and cephalin were the most abundant. The signiflcance of this finding is discussed.     

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.     

Genetic polymorphisms of Echinococcus tapeworms in China as determined by mitochondrial and nuclear DNA sequences

The genetic polymorphisms of Echinococcus spp. in the eastern Tibetan Plateau and the Xinjiang Uyghur Autonomous Region were evaluated by DNA sequencing analyses of genes for mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear elongation factor-1 alpha (ef1a). We collected 68 isolates of Echinococcus granulosus sensu stricto (s.s.) from Xinjiang and 113 isolates of E. granulosus s. s., 49 isolates of Echinococcus multilocularis and 34 isolates of Echinococcus shiquicus from the Tibetan Plateau. The results of molecular identification by mitochondrial and nuclear markers were identical, suggesting the infrequency of introgressive hybridization. A considerable intraspecific variation was detected in mitochondrial cox1 sequences. The parsimonious network of cox1 haplotypes showed star-like features in E. granulosus s. s. and E. multilocularis, but a divergent feature in E. shiquicus. The cox1 neutrality indexes computed by Tajima’s D and Fu’s Fs tests showed high negative values in E. granulosus s. s. and E. multilocularis, indicating significant deviations from neutrality. In contrast, the low positive values of both tests were obtained in E. shiquicus. These results suggest the following hypotheses: (i) recent founder effects arose in E. granulosus and E. multilocularis after introducing particular individuals into the endemic areas by anthropogenic movement or natural migration of host mammals, and (ii) the ancestor of E. shiquicus was segregated into the Tibetan Plateau by colonising alpine mammals and its mitochondrial locus has evolved without bottleneck effects.     

Echinococcus granulosus: secretory activity of the rostellum of the adult cestode in situ in the dog.

Secretary activities associated with the rostellum of adult Echinococcus granulosus were studied using histological, histochemical, and ultrastructural techniques, following rapid fixation of the cestodes in situ in the small intestine of the anaesthetised dog. Studies concentrated on the host-parasite interface from 30 to 35 days postinfection. At this time, contraction of the muscular rostellar pad appeared to be associated with extension of the apical rostellum into a crypt of Lieberkühn. Crypt invasion by the apical rostellum coincided with morphological changes and secretory activity in a group of modified tegumental cells, previously referred to as the rostellar gland. Secretory material, a cystine-rich protein, was observed in the nuclei and cytoplasm of the rostellar gland cells. Release of this material into the interface was seen only following crypt invasion by the apical rostellum. Although the mechanism of release is not clear, it may be analagous to holocrine secretory mechanisms, since apparent degeneration of the rostellar gland region was associated with secretion. Possible functional activities of the secretion associated with hook formation, nutrition, regulation, adhesion, and protection are discussed.     

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.     

Epidemiology and Diagnosis of Echinococcosis in Canada

Two species of Echinococcus occur in Canada: (1) E. multilocularis and (2) E. granulosus. E. multilocularis, originating in the Arctic, is spreading southwards and has reached Saskatchewan and the Dakotas. The original hosts are foxes but dogs and cats are alternatives. The larvae occur in field mice as multilocular microcysts containing numerous protoscolices, but in man the cysts are alveolar and sterile and resemble both in histology and growth a cholangiocellular carcinoma of the liver with metastases. Signs and symptoms are chronic and poorly defined. Diagnosis is difficult. Test antigens are not yet satisfactory. E. granulosus has a sylvatic cycle, the adult tapeworms living in wolves and dogs, while the larvae occur only in Cervidae and man. The cysts occur almost exclusively in the lungs as unilocular, macrocystic, relatively benign tumours, although abnormal complications can occur. The Casoni intradermal sensitivity test, its technique and interpretation are discussed.     

The occurrence of Echinococcus spp. in golden jackal (Canis aureus) in southwestern Hungary: Should we need to rethink its expansion?

Alveolar echinococcosis and cystic echinococcosis are severe zoonotic diseases caused by Echinococcus multilocularis and Echinococcus granulosus s.l. in Europe. To present knowledge, in the European continent, the most important definitive hosts of these parasites belong to the Canidae family. The golden jackal as an opportunistic mesopredator frequently preys on rodents including arvicolids and other easily available food resources, such as viscera and other carrion. By these reasons, the golden jackal can promote the maintenance of both Echinococcus multilocularis and Echinococcus granulosus s.l. Our investigation was conducted in the southwestern part of Hungary where one of the densest golden jackal populations exists. We examined altogether 173 golden jackal small intestines to determine the presence of Echinococcus multilocularis and Echinococcus granulosus s.l. After the molecular diagnostic procedure, we found 27 Echinococcus multilocularis-positive (prevalence: 15.6%; mean intensity: 664 worms) and three Echinococcus granulosus s.l. infected hosts (prevalence: 1.7%; mean intensity: 554.3 worms). We suggest the invasion of the golden jackal in Europe can enhance the spread of both Echinococcus multilocularis and Echinococcus granulosus s.l. This novel epidemiological situation can influence the geographical distribution of these helminths and the characteristics of their endemic in different host species, as well as in humans.     

Lesions induced by complement in vitro on the protoscoleces of Echinococcus multilocularis: a study by electron microscopy.

Changes in the ultrastructure of the tegument of Echinococcus multilocularis protoscoleces during complement-mediated lysis in vitro was studied using transmission and scanning electron microscopy. It was found that the total disintegration of protoscoleces by complement proceeds through formation of ‘tegumental bubbles’ and disruption of the external plasma membrane. This sequence of events was evident in the appearance of numerous loose membrane fragments and vesicles, the lifting of the external unit membrane of the microtriches and the release of organelles from the distal cytoplasm. Subsequent events, such as the appearance of a ‘fuzzy’ coat and disruption of the basement membrane, were probably due to autolysis.     

Characterization of emY162 encoding an immunogenic protein cloned from an adult worm-specific cDNA library of Echinococcus multilocularis

A cDNA library based on mRNA from adult worms of Echinococcus multilocularis was constructed. One cDNA clone, emY162, was isolated from this cDNA library. The putative protein from emY162 cDNA consists of 153 amino acids and has a predicted molecular weight of 17.0 kDa. The amino acid sequences of EMY162 are predicted to be a hydrophobic N-terminus conserving a secretory signal, and a hydrophobic C-terminus encoding a transmembrane domain or glycosyl-phosphatylinositol membrane anchor, and to have single fibronectin type III-like domain. In addition, it was shown that the emY162 gene (1738 bp) in the E. multilocularis genome DNA consists of three exons and two introns, and that emY162 is expressed in all four stages (protoscoleces, cultured metacestodes, immature adult worms and mature adult worms). Moreover, immunity to recombinant EMY162, which comprises the fibronectin type III-like domain on the EMY162 protein, was examined. Immune responses to the recombinant EMY162 were studied by using serum from dogs infected with E. multilocularis. Strong IgG immune responses were detected in Western blots.     

A new intermediate host for Echinococcus multilocularis: The southern red-backed vole (Myodes gapperi) in urban landscape in Calgary,Canada

Human Alveolar Echinococcosis (HAE) is a potentially fatal parasitic disease caused by Echinococcus multilocularis, a cestode characterized by a sylvatic life-cycle involving several species of rodents and lagomorphs as intermediate hosts and canids as definitive hosts. Despite the wide distribution of the parasite in North America, the number of competent intermediate host species identified to date is still relatively small, and mainly includes the northern vole (Microtus oeconomus), brown lemming (Lemmus sibiricus), northern red-backed vole (Myodes rutilus), deer mouse (Peromyscus maniculatus) and meadow vole (Microtus pennsylvanicus).By monitoring the infections in rodents in the city of Calgary (Alberta, Canada), we have detected a case of severe alveolar echinococcosis in a southern red-backed vole (Myodes gapperi), a species never reported before as an intermediate host for this parasite. Observation of protoscolices in the intra-abdominal multilocular cysts indicates that M. gapperi could act as a competent intermediate host for the transmission of E. multilocularis.Since M. gapperi can be found in close proximity to, and within metropolitan areas, this species could play a role in the establishment and maintenance of the sylvatic life-cycle of E. multilocularis in urban landscapes, where the potential for zoonotic transmission is higher. The new intermediate host reported needs to be taken into account in future surveys and transmission models for this parasite.     

Excretory/secretory-products of Echinococcus multilocularis larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro

Background

Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action.

Methodology/Principal Findings

We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naïve T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro.

Conclusions

This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.     

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.     

Primary alveolar echinococcosis: Course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis

We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host–parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.     

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.