Towards delimitation of the Echinococcus multilocularis parasite's southernmost range in France.

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.

High variability in the number of E. multilocularis eggs in cat feces collected in the field.

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.

Soil contamination by Echinococcus multilocularis in rural and urban vegetable gardens in relation to fox, cat and dog faecal deposits.

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.

TITLE: 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. ABSTRACT: 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.

Unravelling the genetic diversity and relatedness of Echinococcus multilocularis isolates in Eurasia using the EmsB microsatellite nuclear marker.

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.

Rodent control programmes can integrate Echinococcus multilocularis surveillance by facilitating parasite genotyping: the case of Arvicola terrestris voles screening in France.

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.

Investigation of Echinococcus multilocularis in foxes and dogs in Pakistan by detection of copro-DNA.

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.

Microsatellite Investigations of Multiple Echinococcus Granulosus Sensu Stricto Cysts in Single Hosts Reveal Different Patterns of Infection Events between Livestock and Humans.

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus sensu lato (s.l.) is a worldwide zoonosis and E. granulosus sensu stricto (s.s.) is the most common species associated with animal and human diseases. The objective of this study was to obtain a better understanding of CE infection in livestock and humans from very low and high endemic areas-France and Tunisia-by studying the genetic diversity of E. granulosus s.s. at the intra-individual host level. This genetic diversity was studied using EgSca6 and EgSca11 microsatellite profiles in 93 sheep from France and Tunisia, and in 12 cattle and 31 children from Tunisia only, all presenting multiple CE cysts (2 to 10 cysts). Overall, 96% of sheep, 92% of cattle, and 48% of children had at least two cysts with different microsatellite profiles. Inversely, 35% of sheep, 17% of cattle, and 65% of children had at least two cysts with the same microsatellite profile. The genotyping results for the CE samples highlight high and similar genetic diversity in France and Tunisia, suggesting that the probability of being successively infected by CE of the same microsatellite profile was rare in both countries. Therefore, our results suggest that in rare cases, several eggs of the same microsatellite profile, from two to seven in our data, can be ingested simultaneously in a single infection event and develop into several cysts in livestock and children. They also indicate that multiple infection events are frequent in livestock, even in a low endemic country such as France, and are less frequent but not negligible in children in a high endemic country such as Tunisia. Moreover, this is the first time that genetic evidence of secondary CE has been found. Further studies are needed to better assess the pattern of infection events in livestock and humans, especially by studying the genetic diversity of adult worms in definitive hosts.

National survey and molecular diagnosis of Echinococcus granulosus sensu lato in livestock in France, 2012.

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.

Asian Admixture in European Echinococcus multilocularis Populations: New Data From Poland Comparing EmsB Microsatellite Analyses and Mitochondrial Sequencing.

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.

Molecular identification of cystic echinococcosis in humans and pigs reveals the presence of both Echinococcus granulosus sensu stricto and Echinococcus canadensis G6/G7 in the hyperendemic focus of the Republic of Moldova.

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.

Investigating the genetic diversity of Echinococcus granulosus sensu stricto with new microsatellites.

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.