Echinococcus multilocularis: identification of proteins inducing antibody formation in metacestode infection

An approach to the identification of parasite proteins which are immunogenic in natural infections is described, using the infection with the larval stage of Echinococcus multilocularis as a parasite model. Metacestode proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose sheets (Western blotting). Subsequently, immune recognition of the proteins was performed with various host sera and antigen-antibody complexes were detected enzymatically. Using homologous antisera, different patterns of immunogenic bands were revealed by sera of different host species. Cross-reactions with sera from individuals infected with unrelated helminths were analysed. Four proteins of E. multilocularis which failed to show any cross-reaction were identified.     

Echinococcus multilocularis laminated-layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro.

Echinococcus multilocularis and Echinococcus granulosus cause alveolar and cystic (unilocular) echinococcosis, respectively, in humans and animals. It is known that these parasites can affect, among other molecules, nitric oxide (NO) production by periparasitic host cells. Nevertheless, detailed dissection of parasite components specifically affecting cell NO production has not been done to date. We compare the effect of E. granulosus and E. multilocularis defined metacestode structural (laminated-layer associated) and metabolic (14-3-3 protein, potentially related with E. multilocularis metacestode tumor-like growth) components on the NO production by rat alveolar macrophages in vitro. Our results showed that none of these antigens could stimulate macrophage NO production in vitro. However, a reversed effect of some Echinococcus antigens on NO in vitro production was found when cells were previously exposed to LPS stimulation. This inhibitory effect was found when E. multilocularis laminated-layer (LL) or cyst wall (CW) soluble components from both species were used. Pre-stimulation of cells with LPS also resulted in a strong, dose-dependent reduction of NO and iNOS mRNA production after incubation of cells with the E14t protein. Thus, the E. multilocularis 14-3-3 protein appears to be one of the components accounting for the suppressive effect of the CW and LL metacestode extracts.     

Evidence for an increasing presence of Echinococcus multilocularis in foxes in The Netherlands

Echinococcus multilocularis, a tapeworm causing alveolar echinococcosis which is considered a serious zoonosis known to affect humans, appears to be expanding its geographical range in Europe. We studied the emergence of the parasite in the European westernmost edge of its geographical distribution, based on two consecutive parasitological examinations of red foxes (Vulpes vulpes) sampled between 1996 and 2003 in The Netherlands. The average worm count increased from 2.6 worms per fox in the first surveillance to 16.6 worms per fox in the second. Using a mathematical model for a spatially spreading parasite, we found a strong indication that the parasite population is increasing in number and is spreading northward at the speed of 2.7 km per year. The reproduction number (R0), reflecting the parasite's transmission process, is estimated from the surveillance data and it is likely to be more than 1 but not exceeding a value of 4. We analysed a parasite control strategy by estimating the critical fox density for parasite elimination. We conclude that E. multilocularis is an emerging parasite in The Netherlands and thus in the western part of Europe. Control will be very difficult given the current high fox population density.     

Uniform strobilar development of Echinococcus multilocularis in vitro from protoscolex to immature stages

The aim of this study was to obtain uniformity in strobilar development of Echinococcus multilocularis from protoscoleces in vitro. The isolate of E. multilocularis used was derived initially from a human case in France and subsequently maintained in the laboratory by intraperitoneal passage in Meriones unguiculatus. Protoscoleces used for culture were obtained using preparative procedures in which parasite tissue was disrupted gently with minimal exposure to pepsin and acidic conditions followed by immediate exposure to pancreatin in alkaline solution. Resultant cultures contained large numbers of evaginated, active, vermiform stages, which exhibited uniform strobilar development with formation of the first proglottid and segment and limited maturation of the first proglottid. All worms that exhibited proglottization subsequently segmented. Further proglottization did not occur and all worms degenerated within a few days following segmentation. The results are discussed in light of current knowledge of the relationships of somatic and germinal processes in Echinococcus. In view of these results, further studies should be encouraged to improve strobilar development of E. multilocularis in vitro.     

Observations on the development of Echinococcus multilocularis in cats

The development of a European isolate of Echinococcus multilocularis was compared in cats and dogs at the end of the prepatent period. Echinococcus multilocularis established in all dogs and cats, but worm recovery was significantly greater from dogs than from cats. Overall, worms in cats were not as advanced as those in dogs in terms of development and maturation, but there was no evidence of retarded development or stunted forms. These results confirm that dogs are highly susceptible to E. multilocularis, whereas cats have lower and more variable recovery rates. However, because cats produce thick-shelled eggs of E. multilocularis after experimental and natural infections, they have to be regarded as potential sources of infection both for intermediate and accidental hosts, including humans. However, their general role in the epidemiology of the infection has yet to be determined.     

Nucleotide Sequences of DNA Fragments of Encephalitozoon cuniculi Amplified by Polymerase Chain Reaction with Primers Regarded as Specific for Echinococcus

Encephalitozoon -like spores were separated from a human echinococcal liver lesion, which was caused by Echinococcus multilocularis. They were found to fall into the species Encephalitozoon cuniculi , which was shown to have En. cunniculi specific DNA by way of polymerase chain reaction (PCR). We also used PCR to genetically discriminate between the En. cuniculi spores and the Ec. multilocularis larvae. Two primer sets, known to be specific for Echinococcus , were examined. These primers were expected to work normally when the two quite different DNA preparations were tested as templates, i.e. only Echinococcus DNA could give a positive signal in the PCR tests. However, it was found that the two Echinococcus -specific primer sets could amplify not only EC. multilocularis DNA, but also En. cuniculi spore DNA. We then tried to determine the order of nucleotides in the Echinococcus -specific primers-amplified En. cuniculi PCR products and compared the determined sequences with those of Ec. multilocularis. The results clearly indicated that sequencing made little difference between En. cuniculi and Ec. multilocularis.     

Modeling the transmission of Echinococcus granulosus and Echinococcus multilocularis in dogs for a high endemic region of the Tibetan plateau

Echinococcus granulosus and Echinococcus multilocularis abundance and prevalence data, for domestic dogs of Shiqu County, Sichuan Province, People's Republic of China, were fitted to mathematical models to evaluate transmission parameters. Abundance models, assuming the presence and absence of immunity, were fit for both E. granulosus and E. multilocularis using Bayesian priors, maximum likelihood, and Monte Carlo sampling techniques. When the models were compared, using the likelihood ratio test for nested models, the model assuming the presence of immunity was the best fit for E. granulosus infection, with a purgation based prevalence of 8% (true prevalence interval of 8-19% based on the sensitivity of purgation) and a mean abundance of 80 parasites per dog, with an average infection pressure of 560 parasites per year. In contrast, the model assuming the absence of immunity was the best fit for E. multilocularis infection, with a purgation based prevalence of 12% (true prevalence interval of 13-33% based on the sensitivity of purgation) and a mean abundance of 131 parasites per dog, with an average infection pressure of 334 or 533 parasites per year assuming a 5 or 3 month parasite life expectancy, respectively. The prevalence data for both parasites was then fit to a set of differential equations modeling the transition between infection states in order to determine number of infectious insults per year. Infection pressure was 0.21, with a 95% credibility interval of 0.12 to 0.41, infections per year for E. granulosus and 0.52, with a 95% credibility interval of 0.29-0.77, infections per year for E. multilocularis assuming a 5 month parasite lifespan or 0.85, with a 95% credibility interval of 0.47-1.25 infections per year, assuming a 3 month E. multilocularis lifespan in dogs.     

New distribution records of Echinococcus multilocularis in the brown lemming from Barrow, Alaska, USA

We identified Echinococcus multilocularis for the first time in brown lemmings (Lemmus trimucronatus) from Barrow, Alaska, USA. Of 467 brown lemmings trapped between 1995 and 2000, two males and two females (0.9%; 95% confidence interval=0.9+/-0.9%) were found to be infected with metacestodes of E. multilocularis. No metacestodes were found in 17 collared lemmings (Dicrostonyx rubricatus) also trapped at Barrow. In humans, E. multilocularis causes alveolar echinococcosis, which is potentially fatal. Knowledge of the distribution of this parasite is important to protect the public health.     

Echinococcus multilocularis identified in Michigan with additional records from Ohio

Echinococcus multilocularis was identified in a coyote in Indiana in January 1990, prompting an investigation of the distribution and prevalence of the parasite in wild canids in Indiana and surrounding states. In 1990-1991, the parasite was found throughout northern and central Indiana, in northwestern Ohio, and in east-central Illinois. In 1993-1994, 162 wild canids (97 red foxes, 54 coyotes, 11 gray foxes) were collected from Michigan, and an additional 75 (55 red foxes, 7 coyotes, 13 gray foxes) from Ohio, and examined for this parasite. Of these, 15 wild canids (6.3%) were found to be infected with E. multilocularis , including 4 of 97 (4.1%) red foxes from Michigan and 9 of 55 (16.4%) red foxes and 2 of 7 (28.6%) coyotes from Ohio. In Michigan, all infected animals were from the central and southwestern parts of the state. No infected animals were found in northern Michigan, including the Upper Peninsula. In Ohio, infected animals were limited to the northwestern and west-central portions of the state. These findings constitute new state and distribution records for E. multilocularis in the midwestern United States and indicate that the parasite continues to spread eastward and into Michigan from the south.     

Immunisation with Salmonella typhimurium-delivered glyceraldehyde-3-phosphate dehydrogenase protects mice against challenge infection with Echinococcus multilocularis eggs

Recombinant glyceraldehyde-3-phosphate dehydrogenase of the cestode parasite Echinococcus multilocularis was expressed in Escherichia coli and in Salmonella typhimurium. The potential of different forms of the recombinant antigen to protect BALB/c mice against oral challenge infections with E. multilocularis eggs was evaluated. Oral or intraperitoneal immunisation with live attenuated S. typhimurium as a carrier for recombinant glyceraldehyde-3-phosphate dehydrogenase of the E. multilocularis resulted in significant protection, reducing the number of developing metacestodes up to 79.8%. The sera of protected animals did not contain detectable amounts of antibody against glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis. By contrast, although anti-glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis antibodies were detectable in the sera, immunisation with E. coli-expressed recombinant glutathione-S-transferase-fusion protein or with glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis fused to a 6HIS-tag failed to protect the animals against oral challenge infections. These data emphasise that antigen delivery systems play a critical role in vaccination and the induction of protective immunity against helminth parasites.     

Milbemycin oxime in a new formulation, combined with praziquantel, does not reduce the efficacy of praziquantel against Echinococcus multilocularis in cats

Twenty European domestic cats were each infected with 15,000 protoscoleces of Echinococcus multilocularis extracted from metacestodes grown in experimentally infected common voles (Microtus arvalis). Sixteen days after infection, ten cats were treated with a broad-spectrum anthelmintic and acaricide comprising praziquantel and milbemycin oxime. Five days later treated and untreated cats were euthanized and the intestine examined for E. multilocularis. Five of ten untreated cats were infected with E. multilocularis with worm burdens ranging from 235 to 1920 worms per cat. No E. multilocularis were recovered from any of the treated cats. This study has demonstrated that this new combination broad spectrum anthelmintic and acaricide for cats is highly efficacious against E. multilocularis and the relevance of this is discussed.     

Alveolar Echinococcus species from Vulpes corsac in Hulunbeier, Inner Mongolia, China, and differential development of the metacestodes in experimental rodents

Adults of alveolar Echinococcus species with different uterine structures were collected from Vulpes corsac in the Hulunbeier Pasture of Northeastern China in 2001. They were Echinococcus multilocularis Leuckart, 1863 (type No. 3, similar to E. m. multilocularis), with vaselike uterus; Echinococcus cf. sibiricensis Rausch et Schiller, 1954 (type No. 1), with pyriform uterus; and Echinococcus sp. (type No. 2) with spherical uterus at segment top. The metacestode development in rodents also differed among those 3 parasites. In the case of E. multilocularis (type No. 3), many germinal cells grew on the inner surface of early cysts, most of which metastasized into host tissue to form brood vesicles or from the germinal cell layer on the inner surface of the vesicle wall. Cells also had an appearance of proliferating by means of alveolar buds from alveolar tissue that developed outward to form new alveolar foci. In Echinococcus cf. sibiricensis (type No. 1), the formation of alveolar vesicles was due to the metastasizing of germinal tissue into host tissue; protoscoleces grew in the center of alveolar vesicles. In type No. 2 (Echinococcus sp.), the formation of the alveolar vesicle was by multiplication of germinal cell layers on the inner surface of alveolar cysts; protoscoleces grew from the germinal cell layer and mesh in the vesicles. On the basis of uterine structure and on differences in development of metacestodes in experimental rodents, we propose that the 3 types of Echinococcus represent 3 independent species: E. multilocularis, Echinococcus sibiricensis, and Echinococcus sp. (type No. 2-as yet under study).     

Echinococcus multilocularis in north Italy

Alveolar echinococcosis is a zoonotic infection caused by the metacestode of the tapeworm Echinococcus multilocularis. Fox populations living in the Alpine regions of Italy had been considered free from this parasite until 2002, when two infected foxes were detected in Bolzano province (Trentino Alto Adige region) near Austrian border. A modified nested PCR analysis was used to detect E. multilocularis DNA in faecal samples belonging to red fox populations from five Italian regions. A total of 522 faecal samples were analysed from foxes shot in Valle d'Aosta (N = 65), Liguria (N = 44), Lombardy (N = 105), Veneto (N = 67), and Trentino Alto Adige (N = 241) regions. Among these, 24 samples, all from the Trentino Alto Adige region, were found positive. Moreoever, 1406 faecal samples of red foxes were analyzed by CA-ELISAs commercial test kit. This paper provides an update of the epidemiological knowledge of this parasite in north Italy.     

In vivo inhibition by ethyloxanilates of alkaline phosphatases of the cestode Echinococcus multilocularis

Alkaline phosphatases (E.C.3.1.3.1), membranous enzymes of the cestode Echinococcus multilocularis have been studied in the parasite and in the experimental host liver. Synthetic inhibitors interaction with metacestode alkaline phosphatases is reported. In regard to the alkaline phosphatases inhibition, the ethyloxanilate 2 is more efficient in the cestode itself than in the host liver.     

Observations on Echinococcus multilocularis in the definitive host

Six dogs were found to be susceptible to experimental infections with a European isolate of Echinococcus multilocularis from southern Germany. Two cats were only poorly susceptible. Adult worms were not evenly distributed throughout the small intestine and the majority of parasites were found in the posterior region. The mode of attachment of E. multilocularis in the dog was similar to that for E. granulosus with the adult worm extending its rostellum deep within a crypt of Lieberkühn. In cats only few worms were found to have penetrated deeply between the villi. E. multilocularis was found to possess a modified group of rostellar tegumental cells, morphologically and functionally identical to those described in E. granulosus and previously referred to as the "rostellar gland". By studying development in vivo and in vitro, the time required for the production of shelled eggs was demonstrated to be only 28 days. Concurrent experimental infections in dogs with E. multilocularis and E. granulosus revealed that both species will develop together in the same host. Their development was not retarded in any way by the presence of the other and both species were able to coexist in the same area of the intestine.     

Comparison of the antigenicity of protoscoleces and microvesicles of Echinococcus multilocularis prepared from rats

Rats are known to be relatively resistant to infection with Echinococcus multilocularis. However, when rats are inoculated with the parasite tissues, E. multilocularis proliferates slowly at first but after 6 months the cysts increase in size considerably and contain large numbers of protoscoleces. As rats survive for 18 months or longer, approximately 100 ml of packed protoscoleces can be produced from each rat. A comparison of the antigenicity of the protoscoleces and microvesicles by immunoblot methods showed that both Em18 and Em16 are shared components between both protoscoleces and microvesicles, although the latter have some additional antigenic components. In antigens prepared from protoscoleces, the banding patterns around Em18 were much simpler than those from microvesicles. Therefore, for serodiagnosis of E. multilocularis, antigens should be carefully prepared from protoscoleces rather than microvesicles from the rat.     

Culture of Echinococcus multilocularis metacestodes: an alternative to animal use

This article reviews the use of an in vitro culture model for the maintenance and proliferation of Echinococcus multilocularis metacestodes and the formation of protoscoleces. This model has been used to identify and characterize parasite molecules involved in host-parasite interactions, and is a suitable tool to perform in vitro drug-screening assays. The development of a simple and easy-to-handle assay to determine the effects of drugs on parasite viability, without the need for time-consuming animal experimentation, has opened the way for larger-scale in vitro drug screening.     

Lethal effects of freezing Echinococcus multilocularis eggs at ultralow temperatures

Methods for killing Echinococcus multilocularis eggs within stool or intestinal samples, without damaging the diagnostic value of the sample, would significantly reduce the risk of animal health providers acquiring alveolar hydatid disease. The first objective of this study was to determine whether E. multilocularis eggs located in fox intestines can survive storage at -70 C for at least 4 days. Results showed that none of 72,000 E. multilocularis eggs remained infectious to defined strains of mice under these conditions, yet, similar eggs recovered from nonfrozen carcasses stored at 4 C for the same time period were viable. The structural identities of adult worms and eggs were not significantly altered by the freezing and thawing processes. These results indicate that ultracold temperatures can be used to kill or inactivate E. multilocularis eggs, making them safe to handle when diagnosing this parasite in definitive hosts. The second objective of this study was to determine whether E. multilocularis eggs could survive freezing to -70 C if commonly used cryopreservation protocols were used. The use of the cryoprotectant solution, 5% dimethyl sulfoxide-35% saline-60% lamb serum, with a -1 C/min freezing rate was unable to prevent the eggs from being killed by freezing to -70 C. Rapid cooling by plunge freezing into liquid nitrogen was also lethal to E. multilocularis eggs. Only a few of the many potential cryopreservation protocols were tested in this study, so it is not yet possible to completely rule out the possibility of preserving these eggs at ultralow temperatures, but it does indicate that temperatures below -70 C are lethal to eggs even under favorable storage conditions.