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.

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 2000's. 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.

Use of FTA® card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa.

Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA® card technology. The DNA samples were taken by applying the FTA® paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA® samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries.

Retrospective analyses of fox feces by real-time PCR to identify new endemic areas of Echinococcus multilocularis in France.

Alveolar echinococcosis is considered to be the most serious zoonosis in the Northern Hemisphere's cold or temperate regions. In Europe, the parasite has a sylvatic life cycle based on predator-prey interactions, mainly between red foxes and small rodents. Echinococcus multilocularis has been observed to have spread across Europe over the last three decades. In France, a westward spread of the parasite's known endemic areas has been described. In this study, a retrospective analysis of fox feces by real-time PCR was carried out in four départements not previously investigated and considered free along with two endemic control departments. The fox feces collected from 2000 to 2004 were analyzed by real-time PCR. Fecal prevalence in the two endemic departments of Doubs and Côte d'Or were estimated at 12 % [6.4-20.0 %] and 4.2 % [1.1-10.3 %], respectively. No positive samples were identified among the 72 feces collected in Drôme or the 112 from Allier, which is consistent with the very low expected prevalence should the parasite be present. Three positive samples were recovered in the Seine-Maritime and Hautes-Alpes départements, resulting in a prevalence of 3.5 % [0.7-10.0 %] and 2.5 % [0.5-7.1 %], respectively. From now on, Hautes-Alpes constitutes the new southern border of the endemic areas in France and confirms the southward expansion previously highlighted. Real-time copro-PCR proved useful in identifying new endemic areas even with low prevalence. Due to the spread of E. multilocularis in France and associated zoonotic risk, it is necessary to expand surveillance in order to fully define all the country's endemic areas. On a continental scale, the development and harmonization of surveillance programs are now needed in order to obtain a global overview of the presence of E. multilocularis and to tailor potential countermeasures.

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.

Echinococcus multilocularis detection in the intestines and feces of free-ranging domestic cats (Felis s. catus) and European wildcats (Felis s. silvestris) from northeastern France.

Experimental studies have demonstrated that cats can be infected by Echinococcus multilocularis, although few data are available concerning their natural infection. This study was designed to compare experimental findings with information on the prevalence of natural E. multilocularis infections of cats in a rural high endemic area. Of 19 intestines of domestic cats (Felis s. catus) and five of European wildcats (Felis s. silvestris) analyzed by segmental sedimentation and counting technique (SSCT), infection by E. multilocularis was observed for one individual of each species, resulting in a prevalence estimated at 5%, (CI95%: 1-26) in domestic cats and at 20% (CI95%: 1-72) in wildcats. High worm burdens (680 and 7040) were noted, but comprised only immature worms. The same EmsB microsatellite profile obtained from the worms' DNA was observed in the two cats as in foxes from the same area and from other European countries. The presence of E. multilocularis DNA was diagnosed in 3.1% (10/321) of the domestic cat feces collected on the field in two villages. However, no E. multilocularis eggs were found after flotation with zinc chloride of the positive feces. The detection of DNA from E. multilocularis was thought to be due to the presence of cells from worms untied from the intestine and corresponding to prepatent infection or due to the digested metacestode. These results from E. multilocularis presence in wild and domestic cat populations agree with those previously obtained by experimental infections. These findings support that these cats play an insignificant role in E. multilocularis transmission, even in a "highly endemic" region. Nevertheless, since the presence of thick-shelled E. multilocularis eggs from cats has already been reported, the associated zoonotic risk cannot be totally ruled out, even if it is very low.

Echinococcus multilocularis infections in dogs from urban and peri-urban areas in France.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease. It is maintained through a sylvatic life cycle based on predator-prey interactions mainly between foxes and rodents. Dogs are also good definitive hosts; and due to their close proximity to humans, they may represent a major risk factor for the occurrence of human cases. In two medium-sized cities of Eastern France (Annemasse and Pontarlier), located in highly endemic areas, 817 dog feces samples were collected and analyzed by a flotation technique followed by a multiplex PCR assay. For the first time in France, we assessed the presence of E. multilocularis DNA in four dog feces samples, in which it represents an estimated prevalence of 0.5% (95% CI; 0.1% <> 1.3%). Eight other samples presented taeniid infections from three different species (Taenia crassiceps, Taenia serialis, and Taenia polyacantha). When considering both E. multilocularis and Taenia sensu lato, prevalence rose to 0.6% in Annemasse and 2.6% in Pontarlier. In this highly endemic context, proper application of the usual deworming recommendations (70% of the dogs were treated twice a year or more) failed to prevent dog infection, particularly for hunting dogs. Our results stressed the need to adapt treatment to the environmental context and to the specific activity of dogs. Further epidemiological surveys in domestic dogs and cats using this coprological approach are still needed to obtain a better overview of infection and the associated zoonotic risk.

Pigs and wild boar in Corsica harbor Echinococcus canadensis G6/7 at levels of concern for public health and local economy.

Cystic echinococcosis (CE) is a parasitic zoonosis widespread in the Mediterranean area. The parasite is commonly maintained in a domestic cycle involving dogs and livestock species. As no new data have been made available for the last 15 years concerning the French Mediterranean island of Corsica, a cross-sectional survey at the slaughterhouse was conducted in 2009-2010 to describe the current presence of Echinococcus granulosus sensu lato in intermediate hosts. Only pig infections with the G6/7 genotype of Echinococcus canadensis were observed. No infection was detected in other breeding species but this should be interpreted with caution because 75% of the cows inspected during the survey were calves, and all sheep and goats were younger than two months old. In parallel four wild boars harvested during the 2010-2011 hunting season were also infected by the same genotype. These data constitute the first report of E. canadensis in France and the first molecular characterization of E. granulosus sensu lato in a wild species in France. The current prevalence observed in pigs (5.9%, n=2527) highlights the fact that CE is still of economic concern on Corsica, an island where certain regional products are produced using pig's liver ("Figatelli"). This prevalence, and the similar one observed in wild boars (4.0%, n=101), is a consequence of certain breeding practices and hunting practices which enable circulation of the parasite in the environment in close contact with humans.

Echinococcus multilocularis in dogs from two French endemic areas: no evidence of infection but hazardous deworming practices.

The life cycle of the zoonotic parasite Echinococcus multilocularis is predominantly sylvatic, involving foxes as definitive hosts infected by predation of rodents, the intermediate hosts. The North-Eastern French departments of Meuse and Haute-Saône are highly endemic, with an estimated fox prevalence of 41% and 36% respectively. Although most of the parasites' biomass occurs in foxes, domestic dogs can also be infected, leading to a major risk of human infection due to the close proximity of dogs and owners. In the present study, dog faeces were collected after praziquantel treatment provided by veterinarians. In all, 860 faecal samples were collected throughout Meuse (n=493) and Haute-Saône (n=367). Intestinal helminth eggs were isolated from the faeces using a flotation technique and observed by microscopy. Parasite species were identified in samples positive for taeniid eggs by sequence analysis after PCR amplification. To study the factors associated with infestation, each sample was linked to a questionnaire filled in by the dog owners. Taeniid eggs were observed in seven faecal samples (0.8%) but none of them were positive for E. multilocularis. Thus, the apparent prevalence of E. multilocularis in dog populations is lower than 1.00% for Haute-Saône and lower than 0.75% for Meuse. In Haute-Saône, a high proportion of dogs observed suspected preying on rodents were not dewormed monthly. In endemic areas, these dogs must be considered at risk of transmitting E. multilocularis to humans.