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Resume Les echinococcoses sont des zoonoses touchant les humains de maniere accidentelle et leur prise en charge implique l’intervention de specialistes issus de nombreuses disciplines. Un consensus a recemment ete obtenu par l’Association mondiale de l’echinococcose sur la standardisation de la terminologie internationale, en anglais. Un groupe de travail francophone multidisciplinaire a ete constitue pour proposer une adaptation specifique a la langue francaise, en prenant en compte les principales regions du monde francophone endemiques pour les echinococcoses. Les principaux changements adoptes sont : 1) la denomination des differentes maladies associees au developpement du metacestode des differentes especes d’echinocoques, « echinococcose kystique », « echinococcose alveolaire » et « echinococcose neotropicale » ; 2) la restriction de l’usage de l’adjectif « hydatique » au stade larvaire du cluster d’especes Echinococcus granulosus sensu lato ; 3) l’harmonisation des expressions qui decrivent la structure des kystes ; 4) la mise en coherence avec la terminologie internationale de la denomination des ex-« vesicules filles », qui sont desormais appelees « kystes filles ». Concernant le traitement chirurgical de l’echinococcose kystique, le systeme de description et l’acronyme « AORC », identique en francais et en anglais pour Abord/Approach, Ouverture/Opening, Resection/Resection, Completude/Completeness, ont ete retenus. L’adoption de cette nouvelle terminologie est essentielle pour la coherence des publications et des ouvrages pedagogiques, et surtout pour une meilleure comprehension de la transmission de ces maladies et une meilleure prise en charge des patients.

International audience; The tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, the most serious parasitic disease for humans in Europe. In Europe, the E. multilocularis lifecycle is based on a prey–predator relationship between the red fox and small rodents. Over the last three decades, the surveillance of E. multilocularis infection in red foxes has led to the description of a wider distribution pattern across Europe. France constitutes the current European western border, but only the north-eastern half of the country is considered endemic. The red fox is the host mainly targeted in E. multilocularis surveillance programmes, but surveys targeting small rodents may be useful for obtaining molecular data, especially when the time-consuming trapping is already carried out in dedicated pest-control programmes. Here, we screened for parasitic lesions in the livers of 1238 Arvicola terrestris voles originating from the historical, but neglected focal area located in central France (Auvergne region) and from Hautes-Alpes, a recently identified endemic department in south-eastern France. This screening identified six voles infected with E. multilocularis in Hautes-Alpes and none in Puy-de-Dôme (Auvergne region) after molecular confirmation. The absence of infected rodents from Puy-de-Dôme can be mainly explained by the generally low prevalence reported in intermediate hosts. The infected Hautes-Alpes samples come all from the same trapping site situated at around 5 km from one of the three fox faecal samples with E. multilocularis DNA collected 15 years prior, thereby confirming the existence and persistence of the E. multilocularis lifecycle in the area. All the rodent E. multilocularis samples from Hautes-Alpes showed the same EmsB microsatellite marker profile. This profile has previously been described in Europe only in the Jura department (central eastern France), located at least 180 km further north. Successive migrations of infected foxes from the historical focal area, including from Jura, to Hautes-Alpes may explain the detection of the parasite in A. terrestris in Hautes-Alpes. Existing trapping efforts in areas where farmers trap A. terrestris for surveillance and pest control can be an effective complement to sampling foxes or fox faeces to obtain E. multilocularis molecular profiles.

Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the Echinococcus genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four Echinococcus species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi, and E. canadensis), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for Echinococcus spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (Taenia solium), and toxocariasis (Toxocara cati and T. canis)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10−4 ng/μL (seven PCR assays positive), corresponding to 37–42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of Echinococcosis species.

International audience; The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe helminthic zoonotic disease distributed in the Northern Hemisphere. The lifecycle of the parasite is mainly sylvatic, involving canid and rodent hosts. The absence of genetic data from most eastern European countries is a major knowledge gap, affecting the study of associations with parasite populations in Western Europe. In this study, EmsB microsatellite genotyping of E. multilocularis was performed to describe the genetic diversity and relatedness of 785 E. multilocularis isolates from four western and nine eastern European countries, as well as from Armenia and the Asian parts of Russia and Turkey. The presence of the same E. multilocularis populations in the Benelux resulting from expansion from the historical Alpine focus can be deduced from the main profiles shared between these countries. All 33 EmsB profiles obtained from 528 samples from the nine eastern European countries belonged to the European clade, except one Asian profile form Ryazan Oblast, Russia. The expansion of E. multilocularis seems to have progressed from the historical Alpine focus through Hungary, Slovakia, the Czech Republic and southern Poland towards Latvia and Estonia. Most of the samples from Asia belong to the Asian clade, with one EmsB profile shared between Armenia and Turkey, and two between Turkey and Russia. However, two European profiles were described from two foxes in Turkey, including one harboring worms from both European and Asian clades. Three EmsB profiles from three Russian samples were associated with the Arctic clade. Two E. multilocularis profiles from rodents from Lake Baikal belonged to the Mongolian clade, described for the first time here using EmsB. Further worldwide studies on the genetic diversity of E. multilocularis using both mitochondrial sequencing and EmsB genotyping are needed to understand the distribution and expansion of the various clades.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis that is considered as the most severe parasitic disease in Europe. The contribution of cat to environmental contamination by E. multilocularis is generally considered as extremely low based on results of experimental infections and worm burden estimations from natural infections. However, the recent collection of numerous cat feces from kitchen gardens in high endemic areas and the detection of E. multilocularis DNA in a significant number of these feces raise the question of the risk of human transmission from cats. This study aimed to provide a quantitative estimation of E. multilocularis eggs in feces from naturally infected cats. A field sampling conducted in 192 kitchen gardens during a joint study led to the collection and analysis of 597 cat feces, among them 7 (1.2%) yielded positive results for E. multilocularis real-time PCR. The entire pellets obtained after homogenization, filtration and centrifugation of a 5 g-sample for each of these 7 feces were examined under a stereoscopic microscope. After assessing their number, 20 taeniid eggs were individually isolated and specifically identified by real-time PCR. Morphologically mature E. multilocularis eggs were identified in 4 samples and the counting of 4 to 43 E. multilocularis eggs per gram in these samples, i.e. 62 to 2331 eggs per feces when the total mass of the feces is considered. The number of eggs counted in 2 feces suggests a biotic potential of some naturally infected cats that largely exceed the previous experimental estimations.

Echinococcus multilocularis eggs are deposited on the ground with the faeces of the carnivore definitive hosts. A reliable assessment of the spatial distribution of E. multilocularis eggs in environments used by humans is crucial for the prevention of alveolar echinococcosis (AE). This study was conducted in 192 rural and 71 urban vegetable gardens in AE endemic areas of north-eastern France. Its objective was to explore the relationship between the spatial distribution of E. multilocularis estimated from the collection and molecular analysis of two types of samples: faeces and soil. A total of 1024 carnivore faeces and 463 soil samples were collected and analysed by real-time PCR. No fox droppings and no positive soil samples were collected from the urban gardens. Positive soil samples, positive carnivore faeces, or both, were found in 42%, 24% and 6% of the sampled rural gardens, respectively. No significant association was found between the detection of E. multilocularis in soil samples collected from 50 gardens during a single sampling session and the extent and frequency of deposits of fox and cat faeces collected during repeated sampling sessions conducted in the previous months. In 19/50 gardens, E. multilocularis was detected in the soil while no positive faeces had been collected in the previous 12 months. Conversely, in 8/50 gardens, no soil samples were positive although positive faeces had been collected in the previous months. Collecting and analysing faeces provide information on soil contamination at a given time, while analysing soil samples provides an overview of long-term contamination.Contamination du sol par Echinococcus multilocularis dans des jardins potagers ruraux et urbains en relation avec les dépôts fécaux de renards, de chats et de chiens.Les œufs d’Echinococcus multilocularis sont déposés sur le sol avec les fèces des carnivores hôtes définitifs. Une évaluation fiable de la distribution spatiale des œufs d’E. multilocularis dans les environnements utilisés par l’homme est cruciale pour la prévention de l’échinococcose alvéolaire (EA). La présente étude a été conduite dans 192 jardins potagers ruraux et 71 jardins potagers urbains des zones endémiques d’EA du nord-est de la France. Son objectif était d’explorer la relation entre la distribution spatiale d’E. multilocularis estimée à partir de la collecte et de l’analyse moléculaire de deux types d’échantillons : des fèces et du sol. Au total, 1024 fèces et 463 échantillons de sol ont été collectés et analysés par PCR en temps réel. Aucun excrément de renard et aucun échantillon de sol positif n’a été collecté dans les jardins urbains. Des échantillons de sol positifs, des fèces de carnivores positives ou les deux ont été trouvés dans 42 %, 24 % et 6 % des jardins ruraux échantillonnés. Aucune association significative n’a été trouvée entre la détection d’E. multilocularis dans les échantillons de sol collectés dans 50 potagers lors d’une unique session d’échantillonnage et l’importance et la fréquence des dépôts de fèces de renards et de chats collectées lors d’échantillonnages répétés conduits au cours des mois précédents. Dans 19/50 potagers, E. multilocularis a été détecté dans le sol alors qu’aucun excrément positif n’avait été collectés dans les 12 mois précédents. A l’inverse, dans 8/50 potagers aucun échantillon de sol n’était positif alors que des fèces positives avait été collectées dans les mois précédents. La collecte et l’analyse de fèces renseignent sur la contamination du sol à un instant donné, alors que l’analyse d’échantillons de sol fournissent un aperçu de la contamination à long terme.

Alveolar echinococcosis is a severe, potentially fatal, parasitic disease caused by ingestion of microscopic eggs of Echinococcus multilocularis. The lifecycle of the parasite is essentially sylvatic, and based on a prey-predator relationship between red foxes and small rodents. A westward expansion from the eastern historical focus has been reported in France, though the parasite has also been detected in the southern Alps. While the focus in the Auvergne region (central France) was described in the 1980s, the southern delimitation of the actual endemic area, especially in the south, was unknown in the absence of dedicated surveys. Red fox samples were collected from 2013 to 2020 in the framework of other transversal epidemiological studies in five sampling areas from southwestern and southeastern France. One hundred and seven intestines were analysed by SSCT, and 221 faecal samples from intestines were analysed by copro-qPCR. None of the 328 foxes exhibited E. multilocularis worms or DNA. Although the presence of E. multilocularis cannot be totally excluded in the departments from the study areas, the sample size tested argues for an absence of the parasite in these studied areas, which is in accordance with the currently known endemic situation in France. These new data are helpful in determining the southernmost limit of E. multilocularis distribution in France. The warm, dry Mediterranean climate in the southeastern areas is less favourable to the transmission of E. multilocularis and especially to the survival of eggs in the environment than the climate in the French Alps or Liguria (Italy) climate where the parasite is present. The intermediate area between the southwestern study areas and the historical focus of Auvergne, which is separated by around 150 km, will be investigated in the coming years. Moreover, an ongoing national surveillance programme on E. multilocularis in foxes is targeting French departements along the edge of the known endemic area both in the southeast and southwest. The data produced will supplement the results of this study, thus greatly helping to define the current distribution of E. multilocularis in France and to target prevention measures to reduce human exposure.

The cestode Echinococcus multilocularis is the causative agent of a severe zoonotic disease: alveolar echinococcosis (AE). The parasite is distributed over a vast area in northern Eurasia and North America, but the impact of AE on human health is highly uneven between different regions. One hypothetical reason for this difference in virulence may be the genetic structure of E. multilocularis which—based on mitochondrial sequences and EmsB microsatellite profiles—forms four distinct clades. These clades correspond approximately to their continents of origin: Asia, Europe, and North America, with a fourth clade apparently restricted to Mongolia and neighboring regions, even though this clade has not yet been described by EmsB genotyping. However, there are various records of genetic variants from the “wrong” region, e.g., “European” haplotypes in Western Canada, which may be the result of introduction or natural migration of host animals. One such example, prompting this study, is the recent record of an “Asian” mitochondrial haplotype in worms from foxes in Poland. At the time, this could not be confirmed by EmsB microsatellite analysis, a method that has proven to possess greater discriminatory power with the E. multilocularis nuclear genome than sequencing of mitochondrial markers. Therefore, worms collected from foxes in Poland were examined both by EmsB analysis and sequencing of the full mitochondrial cox1 gene in order to allocate the samples to the European or Asian cluster. Based on EmsB analyses of 349 worms from 97 Polish red foxes, 92% of the worms clearly showed “European-type” EmsB profiles, but 27 worms (8%) from seven foxes showed profiles that clustered with samples of Asian origin. According to cox1 sequences, a total of 18 worms from 8 foxes belonged to the Asian cluster of haplotypes. The two methods did not fully agree: only 13 worms from three foxes belonged to Asian clusters by both EmsB and cox1, whereas 18 worms from nine foxes belonged to different clusters, according to each marker. Cross-fertilization between worms of Asian origin and those from the European Polish population may explain these conflicting results. The presence of clearly Asian elements in the Polish E. multilocularis population could be the result of introduction of E. multilocularis with host animals (e.g., domestic dogs), or the migration of foxes. In the absence of genetic data from eastern European countries, especially those bordering Poland, it cannot be concluded whether this Asian admixture is typical for a larger area toward central/eastern Europe, or the Polish parasite population is the western extreme of a gradient where both European and Asian elements mingle. Further studies are needed on this subject, preferably using both mitochondrial sequencing and EmsB microsatellite analysis.

International audience; Alveolar echinococcosis (AE) is a zoonosis caused by Echinococcus multilocularis, a heteroxenous parasite belonging to Cestoda class. AE is currently considered an important public health issue, but epidemiological and notably molecular data from several endemic countries, including Pakistan, are sparse. Here we report the first detection of Echinococcus multilocularis in wildlife from Pakistan after real-time PCR and sequencing confirmation in the faecal samples of three foxes from northern Kaghan and Siran regions. The occurrence is estimated at 4.4% (95% CI 0.9-12.4). In order to go further in the epidemiological investigations on E. multilocularis and due to the potential presence of other Echinococcus species, we suggest the need for further epidemiological surveys targeting E. multilocularis and E. granulosus sensu lato isolates from humans and intermediate hosts as well as definitive hosts from wildlife in Pakistan.

For clinical epidemiology specialists, connecting the genetic diversity of Echinococcus multilocularis to sources of infection or particular sites has become somewhat of a holy grail. It is very difficult to trace the infection history of alveolar echinococcosis (AE) patients as there may be an incubation period of five to 15 years before reliable diagnosis. Moreover, the variability of parasitic manifestations in human patients raises the possibility of genetically different isolates of E. multilocularis having different levels of pathogenicity. Thus, the exposure of human patients to different strains or genotypes circulating in geographically different environments may lead to different disease outcomes. Molecular tools, such as the microsatellite marker EmsB, were required to investigate these aspects. This genetic marker was previously tested on a collection of 1211 European field samples predominantly of animal origin, referenced on a publicly available database. In this study, we investigated a panel of 66 metacestode samples (between 1981 and 2019) recovered surgically from 63 patients diagnosed with alveolar echinococcosis originating from four European countries (France, Switzerland, Germany, Belgium). In this study, we identified nine EmsB profiles, five of which were found in patients located in the same areas of France and Switzerland. One profile was detected on both sides of the French-Swiss border, whereas most patients from non-endemic regions clustered together in another profile. EmsB profiles appeared to remain stable over time because similar profiles were detected in patients who underwent surgery recently and patients who underwent surgery some time ago. This study sheds light on possible pathways of contamination in humans, including proximity contamination in some cases, and the dominant contamination profiles in Europe, particularly for extrahepatic lesions.

The parasitic species of the Echinococcus granulosus sensu lato (sl) complex are the causative agents of cystic echinococcosis in humans. The lifecycle of E. granulosus sl is essentially domestic, and is based on the consumption by dogs of hydatid cysts in viscera of livestock species. The aim of this study was to survey E. granulosus sensu lato in livestock in France. A 1-year national survey of E. granulosus sl in livestock at the slaughterhouse was organized in 2012 in France, with systematic molecular confirmation. The prevalence of E. granulosus ss nationally was 0.002% in sheep, mainly focused in the Alpine area, and 0.001% in cattle, with the distribution of cases throughout the country. Echinococcus canadensis G6/7 was observed only in Corsica in pigs, with a prevalence of nearly 1% in the island. A national prevalence of 0.0002% was estimated for E. ortleppi in cattle, due to seven cases distributed in two foci. The results of this survey are of particular interest because of the zoonotic risk associated with the presence of these parasite species, for which systematic control at the slaughterhouse should enable their elimination.

International audience; A greater knowledge of the ecology of the natural foci of tick-borne encephalitis virus (TBEV) is essential to better assess the temporal variations of the risk of tick-borne encephalitis for humans. To describe the seasonal and inter-annual variations of the TBEV-cycle and the epidemiological parameters related to TBEV nymph-to-larva transmission, exposure of small mammals to TBEV, and tick aggregation on small mammals, a longitudinal survey in ticks and small mammals was conducted over a 3-year period in a mountain forest in Alsace, eastern France. TBEV prevalence in questing nymphs was lower in 2013 than in 2012 and 2014, probably because small mammals (Myodes glareolus and Apodemus flavicollis) were more abundant in 2012, which reduced tick aggregation and co-feeding transmission between ticks. The prevalence of TBEV in questing nymphs was higher in autumn than spring. Despite these variations in prevalence, the density of infected questing nymphs was constant over time, leading to a constant risk for humans. The seroprevalence of small mammals was also constant over time, although the proportion of rodents infested with ticks varied between years and seasons. Our results draw attention to the importance of considering the complex relationship between small mammal densities, tick aggregation on small mammals, density of infected questing nymphs, and prevalence of infected nymphs in order to forecast the risk of TBEV for humans.

International audience; Tick-borne encephalitis virus (TBEV) depends mainly on a fragile mode of transmission, the co-feeding between infected nymphs and larvae on rodents, and thus persists under a limited set of biotic and abiotic conditions. If these conditions change, natural TBEV foci might be unstable over time. We conducted a longitudinal study over seven years in a mountain forest in Alsace, Eastern France, located at the western border of known TBEV distribution. The objectives were (i) to monitor the persistence of TBEV circulation between small mammals and ticks and (ii) to discuss the presence of TBEV circulation in relation to the synchronous activity of larvae and nymphs, to the densities of questing nymphs and small mammals, and to potential changes in meteorological conditions and deer densities. Small mammals were trapped five times per year from 2012 to 2018 to collect blood samples and record the presence of feeding ticks, and were then released. Questing nymphs were collected twice a year. Overall, 1344 different small mammals (Myodes glareolus and Apodemus flavicollis) were captured and 2031 serum samples were tested for the presence of antibodies against TBEV using an in-house ELISA. Seropositive rodents (2.1%) were only found from 2012 to 2015, suggesting that the virus disappeared afterwards. In parallel, we observed unusual variations in inter-annual nymph abundance and intra-annual larval activity that could be related to exceptional meteorological conditions. Changes in the densities of questing nymphs and deer associated with the natural stochastic variations in the frequency of contacts between rodents and infected ticks may have contributed to the endemic fadeout of TBEV on the study site. Further studies are needed to assess whether such events occur relatively frequently in the area, which could explain the low human incidence of TBE in Alsace and even in other areas of France.

International audience; In zoonotic infections, the relationships between animals and humans lead to parasitic disease with severity that ranges from mild symptoms to life-threatening conditions. In cities and their surrounding areas, this statement is truer with the overcrowding of the protagonists of the parasites' life cycle. The present study aims to investigate the distribution of a parasite, Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis, using copro-sampling in historically endemic rural settlements of the eastern part of France and in newly endemic areas including urban parks and settlements surrounding Paris. Based on 2741 morphologically identified and geolocalized copro-samples, the density of fox faeces was generally higher in the surrounding settlements, except for one rural area where the faeces were at larger density downtown in the winter. Fox faeces are rare but present in urban parks. Dog faeces are concentrated in the park entrances and in the centre of the settlements. DNA was extracted for 1530 samples that were collected and identified from fox, dog, cat, stone marten and badger carnivore hosts. Echinococcus multilocularis diagnosis and host faecal tests were performed using real-time PCR. We failed to detect the parasite in the surroundings of Paris, but the parasite was found in the foxes, dogs and cats in the rural settlements and their surroundings in the historically endemic area. A spatial structuring of the carnivore stool distribution was highlighted in the present study with high densities of carnivore stools among human occupied areas within some potentially high-risk locations.

The advent of unbiased metagenomic virus discovery has revolutionized studies of virus biodiversity and evolution. Despite this, our knowledge of the virosphere, including in mammalian species, remains limited. We used unbiased metagenomic sequencing to identify RNA viruses in European field voles and rabbits. Accordingly, we identified a number of novel RNA viruses including astrovirus, rotavirus A, picorna-like virus and a morbilli-like paramyxovirus. In addition, we identified a sobemovirus and a novel luteovirus that likely originated from the rabbit diet. These newly discovered viruses were often divergent from those previously described. The novel astrovirus was most closely related to a virus sampled from the rodent-eating European roller bird (Coracias garrulous). PCR screening revealed that the novel morbilli-like paramyxovirus in the UK field vole had a prevalence of approximately 4%, and shared common ancestry with other rodent morbilli-like viruses sampled globally. Two novel rotavirus A sequences were detected in a UK field vole and a French rabbit, the latter with a prevalence of 5%. Finally, a highly divergent picorna-like virus found in the gut of the French rabbit virus was only ~35% similar to an arilivirus at the amino acid level, suggesting the presence of a novel viral genus within the Picornaviridae.

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complexEchinococcus granulosussensu lato. Human infections are most commonly associated withE. granulosussensu stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation withinE. granulosus s.s.G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype.

International audience; A key element to understanding parasite epidemiology is assessing their prevalence in the respective wild reservoir hosts. The tapeworm Echinococcus multilocularis circulates between canid species (definite hosts) and small mammals (mostly rodents; intermediate hosts). Prevalence rates of Echinococcus multilocularis in the intermediate host are most exclusively determined through macroscopic examination of the liver generally followed by molecular or histological diagnostic for parasite species confirmation. The overall objective of the study was to investigate the suitability of Real-Time PCR and Droplet Digital PCR (ddPCR) analysis as tool to detect exposure pressure (frequency of infection events) from E. multilocularis in intermediate hosts even in the absence of macroscopic lesions in the liver. One hundred six small mammals (meadow voles and deer mice) were trapped followed by post-mortem examination including macroscopic evaluation of the liver to detect lesions indicative of infection with Echinococcus multilocularis but also by sampling a piece of liver in absence of lesion to submit it to molecular assay. Macroscopic lesions were present in the livers of two samples. Including the latter two samples, five samples yielded a positive result following Real-Time PCR, whereas 16 samples displayed three or more positive droplets upon ddPCR and were considered positive. Whether these additional cases without macroscopic lesions would have become infectious during the lifespan of the rodent or were abortive or early infections is unclear, but these data suggest levels of exposure of intermediate hosts to the parasite is much higher than assumed.

International audience; The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks.

International audience; Cystic echinococcosis is caused by the parasitic species of the complex Echinococcus granulosus sensu lato. This disease is hyperendemic in the Republic of Moldova. Recent molecular analyses have revealed the exclusive presence of E. granulosus sensu stricto in sheep and cattle. Previous reports of prevalence in pigs suggest the potential presence of Echinococcus canadensis G6/G7, as this species is also reported in neighboring countries. The presence of cystic echinococcosis in pigs was specifically monitored at the slaughterhouse. In the meantime, human cases were genotyped for the first time. E. canadensis G6/G7 was identified in all ten pigs infected by E. granulosus s.l. One human case of infection by E. canadensis G6/G7 was also identified, while E. granulosus sensu stricto was found to be the cause for the 13 others. The description of one human case of E. canadensis G6/G7 has confirmed its zoonotic impact in the country. Future studies will be needed to estimate the relative proportion and distribution of both parasitic species in Moldova.

International audience; Cystic echinococcosis (CE), the parasitic disease caused by the larval stage of Echinococcus granulosus sensu lato (s.l.), is a global public health problem. In Mongolia, despite wide distribution of human CE, not enough information is available on the prevalence and molecular characterization of CE in livestock and its zoonotic linkage with human cases. We investigated the distribution of human CE cases and livestock population using statistical models to get insight into the zoonotic linkage. The incidence of human CE cases increased by a factor of 1.71 for one interquartile range increment in the density of the camel population. No significant association was observed with other livestock species. The samples collected from 96 camels and 15 goats in an endemic region showed a CE prevalence of 19.7% and 6.7%, respectively. All livestock CE were E. granulosus s.l. G6/G7 species of the E. granulosus s.l. complex. The genetic diversity was investigated using the haplotype network based on full cox1 gene analysis of the samples collected from livestock CE and nucleotide sequences previously reported from human CE and wild canids infection in Mongolia. Four haplotypes were identified within the livestock samples, two of which had not been previously reported. A common haplotype was identified among humans, camels, goats, and a wolf, all of which were within the same geographical area. A mixed infection of E. granulosus s.l. G6/G7 with different haplotypes in the intermediate host was identified. To the best of our knowledge, this is the most comprehensive description of the current epidemiological situation of CE in Mongolia with substantial evidence that camels might be the main intermediate host of E. granulosus s.l. G6/G7 in Mongolia. Moreover, our result presents the first report in the country to provide insight into the prevalence of E. granulosus s.l. G6/G7 in livestock.

Human alveolar echinococcosis (AE), caused by the tapeworm Echinococcus multilocularis, is one of the most dangerous zoonoses in the Northern hemisphere. In Europe, the parasite's life cycle is sylvatic, involving small rodents as intermediate hosts and red foxes as the major definitive hosts. Given the severity of this disease in humans and the high levels of environmental contamination with E. multilocularis in endemic areas, it seems crucial to implement control measures in order to prevent human AE. This systematic review identifies previous anthelmintic control programs targeting E. multilocularis in wild and domestic carnivores and evaluates the effectiveness of the different strategies implemented. A search through six databases identified 302 scientific papers for the period 1950–2015, of which only 17 were retained according to the inclusion criteria set. These 17 papers focused on control of E. multilocularis by baiting foxes in highly endemic areas of Europe or Japan, with the exception of one study focused on dogs in Alaska. The papers highlighted differences in baiting types, baiting frequency, choice of control areas and length of treatment period. Overall, these studies resulted in a sharp and statistically significant decrease in parasite prevalence, confirmed by the absence of overlap between confidence intervals for the pooled risk differences of control and treated areas. A monthly baiting frequency was proven to be highly effective at decreasing E. multilocularis prevalence in foxes, even in highly endemic areas and in a short period of time. Nevertheless, when foxes were not fully dewormed, the parasite showed a strong capacity to rapidly recover its initial prevalence. The fox baiting approach appears to be the most useful method for controlling the sylvatic life cycle of E. multilocularis, but it require a cost/benefit analysis before it is likely to be accepted by stakeholders., Highlights • This systematic review evaluated the effectiveness of the control programmes of E. multilocularis. • Differences in baiting types, baiting frequency, selection of control areas and treatment period were highlighted. • A monthly baiting was proved to be efficient to decrease E. multilocularis prevalence in foxes.

Although RNA viruses are likely to exist in every species of cellular life, our knowledge of their biodiversity and evolution has been focused on those that can cause disease in humans and in economically important animals and plants. Recently published studies, however, have shown that every healthy organism can carry viruses. In this study, we present the unbiased discovery of highly divergent RNA viruses in European rodents and rabbits using Next Generation Sequencing (NGS). Tissue samples from different organs were collected from UK rodents and French rabbits and were initially screened for viruses with PCR. Following up on preliminary data, the positive samples were sent for NGS to acquire full genomes and perform unbiased virus discovery. Our findings encompass a number of novel viruses including astrovirus, rotavirus A, hantavirus, picornavirus, coronavirus, paramyxovirus etc. Among the novel viruses, the picornavirus interestingly showed 50 % and 34 % similarity to the closest relative for the non-structural and the structural protein respectively at amino acid level, suggesting a potentially novel genus within the family. This study shows the presence of highly divergent RNA viruses in European rodents and rabbits enriching thus our current knowledge of their origin and evolution. Finally, this study shows that these animal species can be the reservoirs of RNA viruses notorious for cross-species transmission, increasing the risk of a spill-over to humans or livestock.

International audience; Recent surveys at slaughterhouses confirmed the presence of three different species of Echinococcus granulosus sensu lato in France: E. granulosus sensu stricto, E. ortleppi, and E. canadensis G6/7. The latter species was only identified on the French Mediterranean island of Corsica, with a high prevalence in pigs and wild boar. In order to investigate the life cycle of E. canadensis in this region, dog feces were collected in 31 municipalities, mainly from individual kennels. The analysis of fecal samples from 259 dogs by multiplex real-time PCR shows no infection by E. granulosus sensu stricto, but three dogs were infected by E. canadensis G6/7. Genetic analyses of mitochondrial genes (cox1, nad1, nad3, atp6) revealed in two dogs a haplotype previously identified in pigs. The third dog was infected by a new haplotype differing only from the two others from dogs by two mutations in the nad3 gene. This latter haplotype is genetically closer to those identified in pigs rather than those from wild boars. Analysis of questionnaires completed by the owners revealed that the sampled dog population was almost exclusively composed of hunting dogs that had been infrequently dewormed. Most of the owners (78%) leave carcasses of hunter-harvested wild boar in close proximity to their dogs. Nevertheless, genetic results seem to indicate that the three dogs were infected due to their consumption of a pig's infected viscera following home slaughtering. This study confirms the role of dogs as definitive hosts of E. canadensis G6/7 in Corsica. Further molecular studies, notably in human cases, are needed to assess the zoonotic impact of E. canadensis G6/7 in this region.

Tapeworms of the species complex of Echinococcus granulosus sensu lato (s. l.) are the cause of a severe zoonotic disease – cystic echinococcosis, which is listed among the most severe parasitic diseases in humans and is prioritized by the World Health Organization. A stable taxonomy of E. granulosus s. l. is essential to the medical and veterinary communities for accurate and effective communication of the role of different species in this complex on human and animal health. E. granulosus s. l. displays high genetic diversity and has been divided into different species and genotypes. Despite several decades of research, the taxonomy of E. granulosus s. l. has remained controversial, especially the species status of genotypes G6–G10. Here the Bayesian phylogeny based on six nuclear loci (7387 bp in total) demonstrated, with very high support, the clustering of G6/G7 and G8/G10 into two separate clades. According to the evolutionary species concept, G6/G7 and G8/G10 can be regarded as two distinct species. Species differentiation can be attributed to the association with distinct host species, largely separate geographical distribution and low level of cross-fertilization. These factors have limited the gene flow between genotypic groups G6/G7 and G8/G10, resulting in the formation of distinct species. We discuss ecological and epidemiological differences that support the validity of these species.

Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis , two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes , Canis lupus familiaris , Canis lupus , Felis silvestris catus , Meles meles , Martes foina , and Martes martes . With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed ( E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens.

International audience; Baylisascaris procyonis is a zoonotic nematode whose main definitive host is the raccoon, an invasive carnivore in Europe introduced from the United States. B. procyonis causes larva migrans with poor prognosis in humans. This parasite was unexpectedly detected in France for the first time upon molecular screening of wolf faecal samples. Because no patent infection was found, the wolf cannot be considered as a definitive host. This discovery of B. procyonis in France nonetheless raises questions about the parasite status of the expanding raccoon populations in the country, which will be investigated in the future.

International audience; Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the species complex Echinococcus granulosus sensu lato. Within this complex, genotypes G6 and G7 have been frequently associated with human CE worldwide. Previous studies exploring the genetic variability and phylogeography of genotypes G6 and G7 have been based on relatively short mtDNA sequences, and the resolution of these studies has often been low. Moreover, using short sequences, the distinction between G6 and G7 has in some cases remained challenging. The aim here was to sequence complete mitochondrial genomes (mitogenomes) to obtain deeper insight into the genetic diversity, phylogeny and population structure of genotypes G6 and G7. We sequenced complete mitogenomes of 94 samples collected from 15 different countries worldwide. The results demonstrated that (i) genotypes G6 and G7 can be clearly distinguished when mitogenome sequences are used; (ii) G7 is represented by two major haplogroups, G7a and G7b, the latter being specific to islands of Corsica and Sardinia; (iii) intensive animal trade, but also geographical isolation, have likely had the largest impact on shaping the genetic structure and distribution of genotypes G6 and G7. In addition, we found phylogenetically highly divergent haplotype from Mongolia (Gmon), which had a higher affinity to G6.

International audience; Cystic echinococcosis is a zoonotic disease with worldwide distribution caused by the larval stage of the Cestode parasite Echinococcus granulosus sensu lato. Due to the predominance or even the exclusive presence of E. granulosus sensu stricto (s.s.) among E. granulosus species in many areas, the genetic diversity needs to be further investigated at the species level to better understand the inter-and intra-focus epidemiological features. Short sequences of mitochondrial or nuclear genes generally lack or have limited discriminatory power, hindering the detection of polymorphisms to reflect geographically based peculiarities and/or any history of infection. A high discriminatory power can only be reached by sequencing complete or near complete mitogenomes or relatively long nuclear sequences, which is time-consuming and onerous. To overcome this issue, a systematic research for single-locus microsatellites was performed on the nuclear genome of E. granulosus s.s. in order to investigate its intra-species genetic diversity. Two microsatellites, EgSca6 and EgSca11, were selected and characterized. The test of a panel of 75 cystic echinococcosis samples revealed a very high discrimination index of 0.824 for EgSca6, 0.987 for EgSca11, and 0.994 when multiplexing both microsatellites. Testing cystic echinococcosis samples from both liver and lungs in five sheep revealed that these two microsatellites appear to be of particular interest for investigating genetic diversity at the intra-individual host level. As this method has many advantages compared to classical sequencing, the availability of other targets means that it is potentially possible to constitute a panel facilitating large-scale molecular epidemiology studies for E. granulosus s.l.

International audience; Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

International audience; Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

International audience; Among the genotype/species causing cystic echinococcosis, the taxonomic status of Echinococcus canadensis is only partially resolved. Within E. canadensis, four genotypes (G6, G7, G8 and G10) have been described based on short mitochondrial sequences, of which G6 and G7 (the 'camel' and the 'pig' strain, respectively) are closely related and variously regarded as microvariants of a single strain G6/7. Globally, this G6/7 cluster is the second most important agent of human cystic echinococcosis and is the predominant Echinococcus taxon in large parts of sub-Saharan Africa. To add data on the internal structure and the geographical distribution of this cluster, we analysed diversity and population structure of 296 isolates of E. canadensis from sub-Saharan Africa, the Middle East and Europe using the complete mitochondrial cytochrome c oxidase subunit 1 (cox1) (1,608bp) and NADH dehydrogenase subunit 1 (nad1) (894bp) gene sequences. Polymorphism of the mtDNA loci gave 51 (cox1), 33 (nad1) and 73 (cox1-nad1 concatenated) haplotypes. African and Middle Eastern isolates mainly grouped in a star-like structure around a predominant haplotype, while the European isolates produced more diversified networks. Although the cox1 diagnostic sequence for G6 is frequent in the African/Middle Eastern sub-cluster, and that for G7 is common in the European isolates, numerous intermediate variants prevent a clear distinction into 'G6' or 'G7', and the entire taxon is best treated as a common haplotype cluster G6/7. Meanwhile, the G6/7 cluster is clearly distinct from sequences of wildlife isolates of G8 and G10 from the northern hemisphere, and sequences of the latter genotypes were remarkably distant from each other. It is clear from the present study that, based on mitochondrial data, G6/7 is a coherent genotypic entity within E. canadensis that retains substantial intraspecific variance, and sub-populations share common ancestral polymorphisms and haplotypes. This study provides the basis for wider biogeographic comparison and population genetics studies of this taxon.

Despite alarming statistics on cystic echinococcosis (CE) in humans and the importance of dog and ruminant populations, no epidemiological investigations have been performed on CE in Sidi Kacem Province (North-West of Morocco). A part of large research project was devoted to determine the status of CE in ruminants. This paper reports the results of the investigations carried out in the 10 abattoirs of this region, over four successive years (April 2009–March 2013). A total of 1,302 sheep, 652 head of cattle, and 136 goats were postmortem examined and hydatid cysts were collected and examined. The overall CE prevalence of infection was 42.9% in cattle, 11.0% in sheep, and 1.5% in goats. The prevalence shows significant association with age in sheep and cattle; sheep above 3 years and cattle above 5 years are highly infected (64.0% and 72.2%, resp.). Only liver and lungs are found to be infected. Molecular analyses identified G1, G2, and G3 of E. granulosus sensu stricto in liver and lung samples. Cyst fertility was significantly higher in sheep (54.9%) than in cattle (50.3%). These findings suggest that control measures should target not only sheep but also cattle.