Genetic Diversity of the Cestode Echinococcus multilocularis in Red Foxes at a Continental Scale in Europe

Background

Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level.

Methodology/Principal Findings

The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas.

Conclusions/Significance

The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a “mainland–island” system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.     

Infection of red foxes with Echinococcus multilocularis in western Switzerland

In the Jura mountains, Plateau and Alps of western Switzerland important variations in the prevalence of Echinococcus multilocularis infection in red foxes were observed between geographical areas from 1990 to 1995. The Jura mountains and the Plateau had higher mean prevalence levels than the Alps with 30.6, 32.4 and 18.8%, respectively. The highest rate was recorded in the Plateau in the canton of Fribourg with a prevalence of 52.3%. The prevalence of E. multilocularis infection in foxes in the alpine canton of Valais was the lowest (7.1%). Juvenile foxes were found to be more susceptible to E. multilocularis than adults. Adult foxes were less heavily infected in summer and autumn, while the prevalence in juveniles (less than 1 year old) increased between the spring and winter, when they are more than 6 months old. The retrospective data relate to the beginning of the 1990s, since when a drastic prevalence increase of E. multilocularis infection in foxes has occurred in several regions of Europe. Nevertheless, the study is a major contribution to the epidemiological situation of E. multilocularis in central Europe, in that it contains valuable information on spatial distribution and seasonal differences in different age groups of foxes.     

Molecular Identification of Echinococcus multilocularis Infection in Small Mammals from Northeast,Iran

Background

Alveolar echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus multilocularis. Many species of small mammals, including arvicolid rodents or Ochotona spp., are natural intermediate hosts of the cestode. The main aim of this study was to identify natural intermediate hosts of E. multilocularis in Chenaran County, Razavi Khorasan Province, northeastern Iran, where the prevalence of infected wild and domestic carnivores is high.

Methodology/Principal Findings

A program of trapping was carried out in five villages in which this cestode was reported in carnivores. The livers of 85 small mammals were investigated for the presence of E. multilocularis infection using multiplex PCR of mitochondrial genes. Infections were identified in 30 specimens: 23 Microtus transcaspicus, three Ochotona rufescens, two Mus musculus, one Crocidura gmelini, and one Apodemus witherbyi.

Conclusions/Significance

A range of small mammals therefore act as natural intermediate hosts for the transmission of E. multilocularis in Chenaran County, and the prevalence suggested that E. multilocularis infection is endemic in this region. The existence of the life cycle of this potentially lethal cestode in the vicinity of human habitats provides a significant risk of human infection.     

Echinococcus multilocularis: the parasite-host interplay

Alveolar echinococcosis (AE) is a severe chronic helminthic disease caused by the intrahepatic tumor-like growth of the metacestode of Echinococcus multilocularis. Metacestodes are fluid-filled, asexually proliferating vesicles, which are entirely covered by the laminated layer, an acellular carbohydrate-rich surface structure that protects the parasite from immunological and physiological reactions on part of the host. The E. multilocularis metacestode has acquired specific means of manipulating and using the immunological host response to its own advantage. These include the expression of distinct immunoregulatory parasite molecules that manipulate and interfere in the functional activity of macrophages and T cells. Recent research findings have led to a better understanding of the protein- and glycoprotein composition of the laminated layer and the E/S fraction of the metacestode, including Em2- and Em492-antigens, two metacestode antigen fractions that exhibit immunosuppressive or -modulatory properties. Understanding of the events taking place at the host-parasite interface is the key for development of novel immuno-therapeutical and/or chemotherapeutical tools.     

Echinococcus multilocularis in a European beaver from Switzerland

Infection with the larval stage of the cestode parasite Echinococcus multilocularis was diagnosed in a European beaver (Castor fiber) in central Switzerland. The animal was hit, run over by a car, and died of trauma. It was in normal body condition and no signs of disease were seen. At necropsy, multiple cystic structures up to 1 cm in diameter were found in the liver adjacent to the hilus. Within the parasite vesicles, multiple protoscolices were visible. The species was determined to be Echinococcus multilocularis by upon polymerase chain reaction and direct immunofluorescence with MAbG11-FITC. This is the first report of Echinococcus multilocularis in European beaver.     

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.     

Wilderness in the city: the urbanization of Echinococcus multilocularis

A distinct increase in fox populations, particularly in urban areas, has been observed in Europe. This is of particular concern in endemic regions of the small fox tapeworm Echinococcus multilocularis, the aetiological agent of human alveolar echinococcosis. Novel tools have facilitated the investigation of the ecology of urban foxes and have demonstrated the urban wildlife cycle of E. multilocularis. Such studies are essential for estimating the risk of transmission to humans and to determine the basics for the development of control strategies.     

Alkaline phosphatase from Echinococcus multilocularis: purification and characterization.

1. Kinetic and physical parameters of purified alkaline phosphatase from Echinococcus multilocularis metacestodes, livers of infected gerbils and control animals were determined. 2. Km value for p-nitrophenyl phosphate was about 0.05 +/- 0.02 mM for the three enzymes. 3. Vmax values were 357 +/- 67 nmol/min/mg proteins for metacestode enzyme, and 6.7 +/- 1.1 and 6.7 +/- 0.8 nmol/min/mg proteins for liver enzyme of infected and control animals, respectively. 4. Mr and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation of temperature than hepatic enzyme. 6. The isatin inhibition was a competitive inhibition type for parasite and uncompetitive type for host liver enzyme.     

Echinococcus multilocularis in carnivores from the Klatovy district of the Czech Republic

A unique human case of alveolar echinococcosis was described in 1979 from the Klatovy district of the Czech Republic. However, there were no previous epidemiological studies in this area focusing on detection of the source of infection--Echinococcus multilocularis adults producing eggs. During the period June 1997 to April 1999, 29 out of a total of 46 (63.3%) red foxes (Vulpes vulpes) in the Klatovy district and one of four foxes (25.0%) in the Pilsen South district were found to be infected with adult worms of E. multilocularis. No E. multilocularis adults were found in other animals from the Klatovy district (i.e. three specimens of Martes martes, two Martes foina, one Mustela erminea, two Meles meles and one Felis catus f. domestica). An examination of faecal samples from 55 dogs (Canis familiaris) from the Klatovy district resulted in the detection of E. multilocularis DNA in one (1.8%) sample. The present results support the possibility that human alveolar echinococcosis previously described in the Czech Republic had the character of an autochthonous infection. There are also indications of a potential risk of infection to humans.     

Synthetic studies on glycosphingolipids from the parasite Echinococcus multilocularis.

Novel neutral glycosphingolipids isolated from the metacestodes of Echinococcus multilocularis by Persat, may be expected to be involved in host-parasite interactions. We have synthesized these glycosphingolipid analogues containing 2-branched fatty alkyl residues in place of ceramide. The glycosylation of galactosyl donors 4 and 5 with each of the acceptors 2 and 11 in the presence of N-iodosuccinimide (NIS)/TfOH, and the glycosylation of fucosyl donor 13 with acceptors 12 and 20 in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) gave the desired oligosaccharide derivatives at good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycosides 6, 15, 21, and 26 were converted to glycosylimidates 7, 16, 22, and 27, which were condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated give four target glycosphingolipid analogues.     

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.     

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.     

Echinococcus multilocularis: responses to infection in Mongolian gerbils

Mongolian gerbils (Meriones unguiculatus) inoculated intraperitoneally with three acephalic cysts of Echinococcus multilocularis were very susceptible to infection. Aspects of the responses of gerbils to this infection were examined to determine if they could be related to the progress of the infection. Hematologic changes observed during the infection included anemia, reticulocytosis, lymphocytopenia, neutrophilia, monocytosis, and eosinopenia; these changes were related to the size of the infection. Infected gerbils also produced specific protein-A binding antibodies to E. multilocularis. At 14 weeks after inoculation, infected gerbils showed splenomegaly and somewhat elevated serum transaminase levels, although serum 5'-nucleotidase levels were normal.     

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.     

Ecology and epidemiology of Echinococcus multilocularis in Europe

Human alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis has a high mortality rate in untreated patients. The life-cycle of E. multilocularis in Europe predominantly involves foxes as definitive hosts. However, experimental studies demonstrated a comparable biotic potential of E. multilocularis in dogs and raccoon dogs but an insignificant potential in cats. AE occurs in central and eastern Europe at low incidence rates. Recent studies in foxes have shown that E. multilocularis has a wider geographic range (including Italy) than previously thought. In recent years, increases in fox populations have been observed in many European countries, especially in urban areas. As a result, the E. multilocularis cycle is now established in the urban environment. This presents an increased risk of infection for a large human population. Based on these facts and new epidemiological data, possible intervention strategies are presented.     

Echinococcus multilocularis: a rare cestode of the domestic cat in France

A cat living in the city of Annemasse (Haute-Savoie) was parasitized by a single minute specimen of the cestode E. multilocularis. The main characteristics of the parasite are described and illustrated. The cat has not previously been reported as one of the definitive hosts of this cestode in France. A potentially serious epidemiological problem is raised by the presence of an infected cat in an urban estate.