Vanessa Hormaz

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 parasites presence in the park. A comparable environmental contamination with foxs 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.

A flotation/sieving method to detect Echinococcus multilocularis and Toxocara spp. eggs in soil by real-time PCR

Soil can be a source of human infection by many zoonotic helminth species including Echinococcus multilocularis and Toxocara spp. The prevention of alveolar echinococcosis could be greatly improved through the identification of at-risk areas. Yet very few data are available about the detection of E. multilocularis in soil, while more studies have been reported for Toxocara spp. Identification of soil contamination by E. multilocularis eggs requires the use of specific methods. This study describes the development of a method for the detection of E. multilocularis in soil samples with the concentration of eggs using a flotation/sieving method and detection by duplex real-time polymerase chain reaction (PCR). Toxocara spp. egg detection was also undertaken due to the widespread presence of this parasite in soil, despite it being considered less pathogenic. Method sensitivity of 100% was reached for the detection of 10 E. multilocularis eggs spiked in 10 g of soil. Concerning Toxocara spp., method sensitivity was lower but assumed to be due to the reduced effectiveness of the DNA extraction protocol. The parasitological status for E. multilocularis and Toxocara spp. of 63 carnivore fecal samples collected in highly endemic rural areas of France and of soil samples collected under and near these fecal samples was compared. The contamination of soil samples collected under positive fecal samples for E. multilocularis (n = 3) or Toxocara spp. (n = 19) confirmed the transfer of eggs from the definitive host to the environment.

A step forward in the understanding of the presence and expansion of Echinococcus multilocularis in Eastern Europe using microsatellite EmsB genotyping in Poland

Alveolar echinococcosis is a severe zoonotic disease caused by the parasite Echinococcus multilocularis. In Europe, the lifecycle of this cestode is mainly sylvatic based on a prey-predator interaction between the red fox and small rodents as definitive and intermediate hosts, respectively. National surveillance of E. multilocularis in red foxes in Poland has reported a clear distinction between low endemic areas (from 2 to 5.7%) in the western half and high endemic areas (11.8 to 50.0%) in the eastern half of the country. A drastic increase of prevalence has been observed in the eastern half of Poland since the 2000s. Microsatellite EmsB genotyping was performed on 301 E. multilocularis worms from 87 foxes sampled throughout Poland, leading to identification of 29 EmsB profiles. The main profile, Pol19, was identified across the country and accounted for 44.9% of the worms collected. The conformity of 18 Polish profiles was established by comparison with previous profiles identified in Europe, but none corresponded to the most common European profiles. Poland was confirmed as a peripheral area of the main European focus, with more recent colonization by the parasite. The sharing of common profiles mainly by neighboring provinces was confirmed by a clustering analysis identifying four main groups. Expansion of the parasite in Poland in these four groups appears to be influenced by the situation in neighboring countries. Acquiring EmsB genotyping data from eastern European countries, for which very few data are reported, is necessary to understand the expansion of the parasite in the whole of Europe.

EWET: Data collection and interface for the genetic analysis of Echinococcus multilocularis based on EmsB microsatellite

Evolution and dispersion history on Earth of organisms can best be studied through biological markers in molecular epidemiological studies. The biological diversity of the cestode Echinococcus multilocularis was investigated in different cladistic approaches. First the morphological aspects were explored in connection with its ecology. More recently, molecular aspects were investigated to better understand the nature of the variations observed among isolates. The study of the tandemly repeated multilocus microsatellite EmsB allowed us to attain a high genetic diversity level where other classic markers have failed. Since 2006, EmsB data have been collected on specimens from various endemic foci of the parasite in Europe (in historic and newly endemic areas), Asia (China, Japan and Kyrgyzstan), and North America (Canada and Alaska). Biological data on the isolates and metadata were also recorded (e.g. host, geographical location, EmsB analysis, citation in the literature). In order to make available the data set of 1,166 isolates from classic and aberrant domestic and wild animal hosts (larval lesions and adult worms) and from human origin, an open web access interface, developed in PHP, and connected to a PostgreSQL database, was developed in the EmsB Website for the Echinococcus Typing (EWET) project. It allows researchers to access data collection, perform genetic analyses online (e.g. defining the genetic distance between their own samples and the samples in the database), consult distribution maps of EmsB profiles, and record and share their new EmsB genotyping data. In order to standardize the EmsB analyses performed in the different laboratories throughout the world, a calibrator was developed. The final aim of this project was to gather and arrange available data to permit to better understand the dispersion and transmission patterns of the parasite among definitive and intermediate hosts, in order to organize control strategies on the ground.