Epidemiological update of cystic echinococcosis in livestock and assessment of practices related to its control in the Mediterranean area.

Cystic echinococcosis (CE), caused by the tapeworm Echinococcus granulosus, is a zoonotic parasitic disease that still represents a serious threat to human and animal health worldwide. The Mediterranean basin is recognized as one of the major hotspots of CE due to several factors, including the presence of diverse intermediate host species as well as socio-economic and cultural conditions of local communities. This study aims to take a closer look at epidemiological data on CE in the Mediterranean area and assess the knowledge attitudes and practices of shepherds towards this disease in four countries (Algeria, Greece, Italy and Tunisia), highly endemic for CE, with the final goal of identifying highly endemic risk areas and practices in use which might potentially allow the persistence of E. granulosus infection in these areas. To update the epidemiological scenario of CE in Mediterranean areas, a comprehensive review of peer-reviewed literature on CE prevalence data published during the 2017-2023 period was carried out and, through a geographical information system (GIS), a map displaying the current CE distribution in the Mediterranean area was generated. In addition, a questionnaire survey was conducted through in-depth interviews of the farmers to collect information on their management system as well as knowledge attitudes and practices towards CE. From the farmer-participatory survey some risky practices emerged including the non-regular deworming of dogs or the use of ineffective drugs or dosing, as well as the provision of uncooked animal viscera to dogs. Finally, lower levels of knowledge and awareness of the disease was observed among farmers from North Africa compared with those of European countries. In conclusion, the results obtained highlight that CE is still a very serious problem in Mediterranean areas and increased efforts are needed to promote awareness among farmers and to turn research results into policy in order to reduce the spread of this disease, according to the One Health perspective.

Baiting not-owned dogs against Echinococcus granulosus: innovative tools for integrated control.

Cystic echinococcosis (CE), caused by the larval stage of the cestode Echinococcus granulosus, is one of the most widespread zoonoses in Mediterranean countries. Baiting not-owned dogs with praziquantel (PZQ), due to their key role in the maintaining the transmission of CE, currently appears to be the most effective way to limit the transmission of CE, as well as an important aspect to introduce for the control of this parasitic disease. Therefore, this study aims to test 3 types of PZQ-based baits by evaluating different parameters (integrity over time, attractiveness and palatability for dogs, and mechanical resistance after release to different altitudes) and the bait acceptance in field by target animals, i.e. not-owned dogs, by using camera traps. The double PZQ-laced baits (with a double layer of highly palatable chews) showed the greatest resistance in the environment while also preserving the attractiveness and palatability up to 10 days, also withstood heights of 25 m, thus resulting as the most suitable also for drone delivery. The results on the field showed that most of the baits were consumed by not-owned dogs (82.2%), while the remaining were consumed by wild boars (8.9%), foxes (6.7%), badgers (1.1%) and hedgehogs (1.1%), confirming the specific and high attractiveness of the double PZQ-laced baits for the target population and highlights how an anthelmintic baiting programme may be a viable tool for the management of E. granulosus among free-ranging dog populations in endemic rural areas.

An innovative strategy for deworming dogs in Mediterranean areas highly endemic for cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus sensu lato, is a zoonotic parasitic disease of economic and public health importance worldwide, especially in the Mediterranean area. Canids are the main definitive hosts of the adult cestode contaminating the environment with parasite eggs released with feces. In rural and peri-urban areas, the risk of transmission to livestock as well as humans is high because of the free-roaming behavior of owned/not owned dogs. Collecting data on animal movements and behavior using GPS dataloggers could be a milestone to contain the spread of this parasitosis. Thus, this study aims to develop a comprehensive control strategy, focused on deworming a dog population in a pilot area of southern Italy (Campania region) highly endemic for CE. METHODS: Accordingly, five sheep farms, tested to be positive for CE, were selected. In each sheep farm, all shepherd dogs present were treated every 2 months with praziquantel. Furthermore, 15 GPS dataloggers were applied to sheep and dogs, and their movements were tracked for 1 month; the distances that they traveled and their respective home ranges were determined using minimum convex polygon (MCP) analysis with a convex hull geometry as output. RESULTS: The results showed that the mean daily walking distances traveled by sheep and dogs did not significantly differ. Over 90% of the point locations collected by GPS fell within 1500 mt of the farm, and the longest distances were traveled between 10:00 and 17:00. In all the sheep farms monitored, the area traversed by the animals during their daily activities showed an extension of < 250 hectares. Based on the home range of the animals, the area with the highest risk of access from canids (minimum safe convex polygon) was estimated around the centroid of each farm, and a potential scheme for the delivery of praziquantel-laced baits for the treatment of not owned dogs gravitating around the grazing area was designed. CONCLUSIONS: This study documents the usefulness of geospatial technology in supporting parasite control strategies to reduce disease transmission.

Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

[Hydatid cyst sampling by FTA Card method for characterizing Echinococcus granulosus sensu lato in Algeria. Preliminary results]. / Prélèvement des kystes hydatiques par la méthode FTA Card pour la caractérisation moléculaire d'Echinococcus granulosus sensu lato en Algérie. Résultats préliminaires.

Introduction and objectives: Cystic echinococcosis is highly endemic in Algeria and constitutes a major socio-economic problem. Typing the species of the Echinococcus granulosus sensu lato complex circulating in cattle requires the use of a hydatid cyst sampling method adapted to difficult field conditions (high heat and humidity, long transport time). The FTA Card method currently constitutes an effective means of preserving biological samples before their molecular analysis. In the present study, the FTA Card method was used in the collection of hydatid cysts to identify the species of E. granulosus sensu lato circulating in ruminants (intermediate hosts) in eastern Algeria. Material and methods: A PCR was carried out for 41 samples of hydatid cysts taken from six slaughterhouses in eastern Algeria, targeting the cox1 mitochondrial gene. PCR products were visualized by electrophoresis in a 1% agarose gel. Results and conclusion: The results of the molecular analysis of all hydatid cyst samples confirmed the presence of E. granulosus sensu stricto in sheep, cattle and camels. The ubiquitous nature of the G1 genotype has been demonstrated. The use of FTA Card sampling is an efficient and simple method to obtain a biological sample in order to characterize the species of E. granulosus sensu lato in Algeria. The good preservation of the DNA in this matrix will make it easier to obtain new molecular data from difficult regions. The identification of the species of the E. granulosus sensu lato complex involved in the biological cycle is an essential prerequisite for the implementation of control measures, since different host species participate in their evolutionary cycle. The characterization of E. granulosus genotypes is essential to define an appropriate control strategy against cystic echinococcosis.

Cryptic species Hydatigera kamiyai and other taeniid metacestodes in the populations of small mammals in Serbia.

BACKGROUND: Hydatigera (Cestoda: Taeniidae) is a recently resurrected genus with the description of a new species, Hydatigera kamiyai, a cryptic entity within the Hydatigera taeniaeformis species complex. Rodents are intermediate hosts and correct taxonomic identification of H. taeniaeformis sensu lato (s.l.) species is difficult without the use of molecular methods. The aim of this study was to identify and explore the genetic diversity of Hydatigera and other taeniid species. METHODS: Ten different small mammals species (856 individuals) (Rattus rattus, three Apodemus, three Arvicolinae and three Soricidae species) were examined from 2013 to 2023. Captured animals were visually examined for cysts and visible lesions. Two markers were used for amplification and sequencing: cox1 and 12S rDNA. RESULTS: Molecular analysis of cysts and visible lesions revealed four taeniid species: Hydatigera kamiyai, H. taeniaeformis sensu stricto (s.s.), Taenia martis and T. crassiceps. Hydatigera kamiyai was found in Apodemus flavicollis, A. agrarius, Microtus arvalis and Crocidrua leucodon, while H. taeniaeformis s.s. is registered in R. rattus. Hydatigera kamiyai cox1 sequences clustered with European populations and showed at least 25 nucleotid differences compared to Asian, African, Australian and one of our isolates of H. taeniaeformis s.s acquired from a rat, followed by large sequence distances (9.4% to 12.9%), indicating clear molecular distinction of two species. CONCLUSIONS: This is one of the few mitochondrial gene-based studies performed after the description of cryptic entities within the Hydatigera taeniaeformis s.l. complex and represents a valuable contribution to understanding of genetic diversity, host suitability and geographic distribution of these tapeworm species. Also, our study provides an important basis of molecular data from this part of Europe for further studies. We emphasize the importance of additional studies of intermediate hosts, especially rats from Europe and Apodemus spp. and voles from Asia and Africa.

It's a small world for parasites: evidence supporting the North American invasion of European Echinococcus multilocularis.

Echinococcus multilocularis (Em), the causative agent of human alveolar echinococcosis (AE), is present in the Holarctic region, and several genetic variants deem to have differential infectivity and pathogenicity. An unprecedented outbreak of human AE cases in Western Canada infected with a European-like strain circulating in wild hosts warranted assessment of whether this strain was derived from a recent invasion or was endemic but undetected. Using nuclear and mitochondrial markers, we investigated the genetic diversity of Em in wild coyotes and red foxes from Western Canada, compared the genetic variants identified to global isolates and assessed their spatial distribution to infer possible invasion dynamics. Genetic variants from Western Canada were closely related to the original European clade, with lesser genetic diversity than that expected for a long-established strain and spatial genetic discontinuities within the study area, supporting the hypothesis of a relatively recent invasion with various founder events.

Real-time multiplex PCR for human echinococcosis and differential diagnosis.

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.

Title: PCR multiplex en temps-réel pour le diagnostic de l'échinococcose humaine et diagnostic différentiel. Abstract: L'identification moléculaire des pathogènes infectieux humains rares semble être l'une des méthodes actuelles les plus pertinentes pour un diagnostic et une prise en charge rapides des patients. Les techniques de PCR, en particulier la PCR quantitative en temps réel, sont bien adaptées à la détection d'ADN de pathogènes, même pour de faibles concentrations. Les infections à échinocoque sont dues à des helminthes du genre Echinococcus, des espèces étroitement apparentées, impliquées dans des lésions parasitaires affectant les animaux et accidentellement l'homme. Une qPCR multiplex (MLX qPCR), permettant la détection de quatre espèces d'Echinococcus impliquées en Europe dans l'échinococcose alvéolaire (EA) et kystique (EK) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi et E. canadensis), basée sur de courtes cibles mitochondriales a été développée ici. Une collection a été constituée de 81 tissus frais ou fixés en paraffine (FFPE) de lésions d'EA et EK. Les essais de qPCR ont été réalisées en triplex pour la détection d'Echinococcus spp., associés à une qPCR de contrôle d'inhibition. Une PCR duplex a été développée pour le diagnostic de deux autres helminthiases en impasse chez l'Homme (cysticercose (Taenia solium), et toxocarose (Toxocara cati et T. canis). La sensibilité de la qPCR a été évaluée et s'échelonne de 1 à 5 × 10−4 ng/µl (sept essais de qPCR positifs), correspondant à 37 à 42 cycles pour l'ADN quantifiable. La spécificité était de 100 % pour toutes les cibles. Cette qPCR multiplex, adaptée à de faibles quantités d'ADN peut être mise en œuvre au laboratoire pour un diagnostic moléculaire rapide des espèces d'Echinococcus.

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.

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.

Species and genotypes belonging to Echinococcus granulosus sensu lato complex causing human cystic echinococcosis in Europe (2000-2021): a systematic review.

BACKGROUND: This study aimed to fill a gap of knowledge by providing a quantitative measure of molecularly identified species and genotypes belonging to Echinococcus granulosus sensu lato (s.l.) causing human cystic echinococcosis (CE) in Europe during the period 2000-2021. As these species and genotypes are characterized by genetic, animal host and geographical differences, studying the E. granulosus s.l. complex is epidemiologically relevant. METHODS: A systematic review (SR) was conducted on the basis of both scientific and grey literature considering primary studies between 2000 and 2021 in four databases. From a total of 1643 scientific papers, 51 records were included in the SR. The main inclusion criterion for this study was the molecular confirmation of E. granulosus s.l. at the genotype/species level as a causative agent of human CE cases in selected European countries. RESULTS: Relevant data were obtained from 29 out of 39 eligible European countries. This SR identified 599 human molecularly confirmed echinococcal cysts: 460 (76.8%) identified as E. granulosus sensu stricto (s.s.), 130 (21.7%) as E. canadensis cluster (G6/7 and G10), 7 (1.2%) as E. ortleppi (G5), and 2 as E. vogeli (0.3%). Three geographical hotspots of human CE caused by different species of the E. granulosus s.l. complex were identified: (1) E. granulosus s.s. in Southern and South-eastern Europe (European-Mediterranean and Balkan countries); (2) E. canadensis (G6/7) in Central and Eastern Europe; (3) E. ortleppi in Central and Western Europe. This SR also identified data gaps that prevented a better definition of the geographical distribution of the Echinococcus granulosus s.l. species complex in Europe: western Balkan countries, part of Central Europe, and Baltic countries. CONCLUSIONS: These results mandate longitudinal, multi-centre, intersectoral and transdisciplinary studies which consider both molecular and clinical epidemiology in animals and humans. Such studies would be valuable for a better understanding of the transmission of the E. granulosus s.l. species complex and their potential clinical impact on humans.

Droplet digital PCR as a sensitive tool to assess exposure pressure from Echinococcus multilocularis in intermediate hosts.

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.

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.

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.

Genotyping Echinococcus multilocularis in Human Alveolar Echinococcosis Patients: An EmsB Microsatellite Analysis.

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.

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.

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.

Evidence for camels (Camelus bactrianus) as the main intermediate host of Echinococcus granulosus sensu lato G6/G7 in Mongolia.

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.

Analysis of nad2 and nad5 enables reliable identification of genotypes G6 and G7 within the species complex Echinococcus granulosus sensu lato.

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.