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.     

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.     

Modified sugar centrifugal flotation technique for recovering Echinococcus multilocularis eggs from soil

Among soil-transmitted parasitic diseases, alveolar hydatidosis due to the ingestion of Echinococcus multilocularis eggs is becoming a serious problem in Hokkaido, the northern most island of Japan. Dissemination of the infection far from the endemic areas can occur if motor vehicles transmit soil contaminated with eggs. No appropriate and validated method for recovering the taeniid eggs from soil is available. A modified sugar centrifugal flotation technique, using a sucrose solution of specific gravity 1.27 and 0.05% Tween-80, was evaluated as a method to successfully recover eggs from soil. Contamination levels as low as 10 eggs per gram could be detected. This method may be useful to determine the prevalence of E. multilocularis, its transmission, and the potential for by monitoring soil contamination with eggs.     

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.     

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.     

Coproantigen survey for Echinococcus multilocularis prevalence of red foxes in Hokkaido, Japan

An epidemiological survey was conducted on the seasonal variation of Echinococcus multilocularis prevalence in red foxes from 1997 to 1998, using a monoclonal antibody-based detection of the tapeworm coproantigen. Thirty-six breeding dens of reproductive fox families were identified in the endemic area of Koshimizu, eastern Hokkaido, Japan. Fecal samples from each site were examined by coproantigen detection assay and fecal egg examination. Whereas the prevalence of coproantigen positive feces showed no seasonal fluctuation (51.6-66.7%), variation was found in the prevalence of egg positive feces in which a higher prevalence was observed in the summer and winter (31.1 and 38.7%) than spring and autumn (13.3 and 13.5%). Significant differences were observed between juveniles and adult foxes in both examinations. Samples from juvenile foxes gave higher coproantigen positive results and taeniid egg intensity. Those results suggest more juveniles infected with the cestode than adults in the same period. The practical use of coproantigen assay as a survey tool and factors which affect the prevalence and host age-related difference are discussed.     

Sensitivity comparison between Mini-FLOTAC and conventional techniques for the detection of Echinococcus multilocularis eggs

Canines serve as the definitive host of Echinococcus multilocularis. This study evaluated the sensitivity of the Mini-FLOTAC technique (MF) for the detection of E. multilocularis eggs in definitive hosts. First, we investigated the effects of heat inactivation and preservative conditions on the detection rate of eggs obtained from experimentally infected dogs. The sensitivity of MF was compared with that of eight other techniques: the centrifugal flotation with sucrose or zinc sulfate, MGL, AMS III, and a combination of MF and flotation/sedimentation techniques. Finally, we compared the sensitivity of MF and the centrifugal flotation with sucrose for the feces of E. multilocularis-infected foxes. The detection rate reached a plateau level with a specific gravity (s.g.) 1.22 for fresh eggs, but the highest rates were obtained with s.g. greater than 1.32 for heat-inactivated eggs. There was no significant difference in the detection rate among the preservative conditions. MF showed significantly higher EPG than the other techniques. Moreover, it showed higher diagnostic sensitivity for the fox feces than the centrifugal flotation technique. These results suggest that heat inactivation may alter s.g. of E. multilocularis eggs and that MF with zinc sulfate (s.g. = 1.32) would be effective for detecting heat-inactivated E. multilocularis eggs.     

Surveillance and management of Echinococcus multilocularis in a wildlife park

The fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease that may be fatal if untreated. A broad spectrum of mammalian species may be accidentally infected even in captivity. In April 2011, liver lesions due to E. multilocularis were observed during the necropsy of a captive-born nutria (Myocastor coypus) in a French wildlife park, leading to initiation of a study to survey the parasite's presence in the park. A comparable environmental contamination with fox's feces infected by E. multilocularis was reported inside (17.8%) and outside (20.6%) the park. E. multilocularis worms were found in the intestines of three of the five roaming foxes shot in the park. Coprological analyses of potential definitive hosts in captivity (fox, lynx, wildcat, genet, wolf, bear and raccoon) revealed infection in one Eurasian wolf. Voles trapped inside the park also had a high prevalence of 5.3%. After diagnosis of alveolar echinococcosis in a Lemur catta during necropsy, four other cases in L. catta were detected by a combination of ultrasound and serology. These animals were treated twice daily with albendazole. The systematic massive metacestode development and numerous protoscoleces in L. catta confirmed their particular sensitivity to E. multilocularis infection. The autochthonous origin of the infection in all the captive animals infected was genetically confirmed by EmsB microsatellite analysis. Preventive measures were implemented to avoid the presence of roaming foxes, contact with potential definitive hosts and contaminated food sources for potential intermediate hosts.     

Natural Echinococcus multilocularis infection in a Norway rat, Rattus norvegicus, in southern Hokkaido, Japan.

Forty-two rats, Rattus norvegicus, captured at a garbage dump in southern Hokkaido, Japan, were examined, and one was found to be infected with Echinococcus multilocularis. The lesions were found in the liver, lung, mesenteric lymph nodes, greater omentum and also free in the abdominal cavity. No necrosis was observed in any of the lesions, and inflammatory reactions were mild. Protoscoleces were observed in the large liver cysts. A homogenate of these cysts, when transplanted into the abdominal cavity of three Mongolian gerbils and a rat, yielded numerous fully developed protoscoleces at 4-7 months post-inoculation. Judging from this, it is postulated that the rat could become a natural intermediate host for E. multilocularis in this area.     

Experimental studies on Echinococcus multilocularis in Japan, focusing on biohazardous stages of the parasite

Alveolar echinococcosis (AE), caused by the active growth of larval Echinococcus multilocularis mostly in the liver, is usually fatal zoonotic disease if not adequately treated. Humans become infected via oral ingestion of the parasite eggs, which are thus biohazardous to humans and should be handled under restricted conditions. In this review, we present findings in experimental studies mainly performed at a safety facility in Japan, examining the biohazadous stages of the parasite (Hokkaido isolate) including its egg and adult worm stages. This article deals mainly with the parasite development in various experimental and wild animals, environmental factors affecting viability of the parasite eggs, and molecular biological studies on adult worms. The findings shown herein have provided a basis to better understand basic biology and natural transmission of E. multilocularis in Hokkaido, a highly endemic area of AE in northern Japan, and also to establish effective preventive measures against the disease.     

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificites of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions.     

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.     

The impact of globalisation on the distribution of Echinococcus multilocularis

In the past three decades, Echinococcus multilocularis, the cause of human alveolar echinococcosis, has been reported in several new countries both in definitive hosts (canids) as well as in people. Unless treated, infection with this cestode in people is fatal. In previously endemic countries throughout the Northern Hemisphere, geographic ranges and human and animal prevalence levels seem to be increasing. Anthropogenic influences, including increased globalisation of animals and animal products, and altered human/animal interfaces are thought to play a vital role in the global emergence of this pathogenic cestode. Molecular epidemiological techniques are a useful tool for detecting and tracing introductions, and differentiating these from range expansions.     

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.     

A major Echinococcus multilocularis antigen is a mucin-type glycoprotein.

The metacestode of Echinococcus multilocularis is surrounded by a carbohydrate-rich laminated layer, which plays a key role in the establishment of the infection in the mammalian host. A major component of the laminated layer is an antigen referred to as Em2(G11). This highly species-specific antigen has been used for serodiagnoses of alveolar echinococcosis and is suggested to contain carbohydrates as major constituents. The results of this work have shown that immunoaffinity-purified Em2(G11) subjected to size-exclusion chromatography eluted mainly in the void volume, indicating a high molecular weight structure of this antigen. Amino acid analysis revealed a large proportion of threonine and proline residues in Em2(G11). The carbohydrate moiety of the antigen was found to be composed of galactose, N-acetylgalactosamine, and N-acetylglucosamine with a ratio of 2.4:1.0:0.5 as determined by gas-chromatography/mass spectrometry. An isotope tag was introduced to the beta-eliminated glycans, and an integrated mass spectrometric O-glycan profiling and sequencing approach was employed to obtain detailed sequence and linkage information of the unseparated glycoform pool. Novel glycoforms containing mucin-type core Gal1-3GalNAc and branched core structures attached to both serine and threonine residues are described. The data presented reveal that the Em2(G11) antigen is a mucin-type glycosylated protein.     

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.