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

https://doi.org/10.1016/j.meegid.2021.104863Get rights and content

Highlights

  • EmsB genotyping of 785 E. multilocularis samples from Eurasia.

  • Presence of an Asian EmsB profile in Ryazan Oblast, European part of Russia.

  • Expansion of European E. multilocularis population from historical focus to Russia.

  • All four clades of E. multilocularis were identified by EmsB in Russia.

  • Presence of both European and Asian clades of E. multilocularis in Turkey.

Abstract

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.

Introduction

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE) and is distributed in the Northern Hemisphere. AE is considered a severe helminthic zoonosis with mortality of over 90% in untreated or inadequately treated cases within 10–15 years of diagnosis (Wilson et al., 1992). Humans are dead-end hosts and are infected via oral ingestion of E. multilocularis eggs. The lifecycle of the parasite is essentially sylvatic, especially in Europe. In non-Arctic parts of Europe, the red fox (Vulpes vulpes) is the main definitive host, while the parasite also occurs in other carnivores including the raccoon dog (Nyctereutes procyonoides), wolf (Canis lupus), golden jackal (Canis aureus) and Arctic fox (Vulpes lagopus), but also in the domestic dog (Canis lupus familiaris). Intestinal infection of definitive hosts results in the release of eggs into the environment via feces. The main intermediate hosts are voles (Microtus arvalis and Arvicola spp.), which become infected via ingestion of parasite eggs and develop the larval stage of the parasite, commonly in the liver. The lifecycle is completed when a competent intermediate host preys upon an infectious prey (i.e. with protoscoleces). Like in the case of animal intermediate hosts, primary human infection is almost exclusively in the liver, with metacestodes potentially spreading to others organs (Brunetti et al., 2010). The disease's long asymptomatic period of around 5–15 years makes it difficult to understand transmission pathways and associated risk factors (Conraths et al., 2017). In 2010, it was estimated that there were approximately 18,235 (CIs 11,900–28,200) new cases of human AE per annum worldwide, of which 16,629 (91%) occurred in China (Torgerson et al., 2010). Concerning Europe, 168 (0.9%) human AE cases per annum were estimated, of which 110 and 58 from western and eastern European countries, respectively (Torgerson et al., 2010). Russia has the second highest number of reported cases outside China, with 1180 (73%) human AE cases. Turkey is affected to a lesser extent with approximately 100 cases yearly.

The known historical focus of AE in Europe was initially restricted to the German and Swiss Jura, and the German, Austrian and French Alps before the end of 19th century, based on reports of human AE cases (Vuitton et al., 2015). The geographical distribution of human AE in Western Europe had apparently remained stable for many decades until the 1990s, when E. multilocularis infections were first reported in others areas and others countries. However, it is uncertain whether these new areas of infection are the result of recent expansion of the parasite or are rather due to the absence or low level of surveillance efforts, particularly in humans, before the widespread use of imaging techniques. In Western Europe, expansion of the known endemic areas was identified in western France, northern Germany, Luxembourg, Belgium, the Netherlands and northern Italy (Deplazes et al., 2017). In the North of continental Europe, the parasite has so far been identified only in Denmark (Saeed et al., 2006) and southern Sweden (Osterman-Lind et al., 2011), although the arctic Svalbard archipelago (Norwegian territory) had been recognized as an endemic area (Henttonen et al., 2001). In eastern European countries, the epidemiological picture in the Baltic region was recently reviewed by (Marcinkutė et al., 2015) who confirmed that the distribution of E. multilocularis was wider and more frequent in canids hosts than previously anticipated. In Estonia, it was found in both rural (Laurimaa et al., 2016; Laurimaa et al., 2015b; Moks et al., 2005) and urban areas (Laurimaa et al., 2015a), whereas in Latvia and Lithuania to date only in rural areas (Bagrade et al., 2008; Bruzinskaite-Schmidhalter et al., 2012). After the first report in 1994 in Poland (Malczewski et al., 1995), the prevalence of E. multilocularis in red foxes has also increased considerably in the country, reaching values of 50% in some areas (Karamon et al., 2014). The presence of the parasite was also reported from the Czech Republic, Slovakia, Hungary, and Serbia, except that the prevalence observed in red foxes is overall around 30% (Kolářová et al., 2017; Miljević et al., 2019; Miterpáková and Dubinský, 2011; Tolnai et al., 2013). An even lower endemic level was reported in Romania, Slovenia and Croatia (Dušek et al., 2020; Logar et al., 2007; Siko et al., 2011). Data from Belarus, Ukraine and Moldova are still scarce, despite the first descriptions more than 50 years ago (Deplazes et al., 2017). Russia and Turkey both have European and Asian parts, all of which are known to be endemic (Deplazes et al., 2017) and they are also considered two important human AE foci (Baumann et al., 2019; Torgerson et al., 2010).

As the period of detection of the parasite in each country is unlikely to reflect the true period of expansion to these areas, analyzing the genetic diversity of E. multilocularis can provide data to better understand the pathways and the history of expansion among different countries. The sequencing of mitochondrial genes (cox1, cob, nad2) was used to obtain concatenated sequences allowing the identification of four clades, approximately corresponding to the regions of origin of the samples and referred to as the European, North American, Asian and Mongolian clades (Nakao et al., 2009). In Europe, EmsB microsatellite analysis has frequently been used to investigate the genetic diversity of E. multilocularis from local to continental scales (Knapp et al., 2007; Knapp et al., 2007; Knapp et al., 2009). The high discriminatory power of the EmsB microsatellite has shown potential to uncover low genetic diversity, e.g. in areas where the parasite was recently introduced. Using this high-resolution molecular tool, high genetic diversity was observed in the historically endemic areas of the northern Alpine region (the central core area) compared to surrounding eastern and northern European regions (Knapp et al., 2009). Most EmsB profiles (60%) were shared between the central core and peripheral areas, with higher genetic diversity in the central core, arguing for an expansion history of the parasite governed by a “mainland-island” system ruled by founder events. The expansion of the parasite can be explained by the dispersal movement of fox populations. Using EmsB in a national context, a spatiotemporal scenario of the expansion of E. multilocularis could be proposed, with gradual spread from the historical focus in eastern France to the north and the west of the country (Umhang et al., 2014). The identification of already reported EmsB profiles of E. multilocularis isolates from Sweden and Denmark suggests that these countries were recently colonized from the historical European focus towards northern Europe, as peripheral areas in the same transmission system (Knapp et al., 2019). In the island-based geographical context of the High Arctic Svalbard archipelago, the European origin of the parasite was excluded as the EmsB profiles clustered with North America samples (i.e. Alaska, Saint Lawrence Island) in the Arctic group (Knapp et al., 2012). Hungary and Poland were also identified as peripheral areas of the historical European focus, sharing the same EmsB profiles with neighboring countries (Casulli et al., 2010; Umhang et al., 2017). Nevertheless, admixture of E. multilocularis lineages typical for Asia was detected in Poland, using both mitochondrial and EmsB markers (Karamon et al., 2017; Umhang et al., 2021). These 31 worms from 12 foxes that contained ‘Asian’ sequences or profiles originated from eastern and mainly western Poland. The absence of genetic data from other eastern European countries made it impossible to draw conclusions on whether these worms in Poland are the result of long-distance introduction events, or whether they were the westernmost representatives of a continuous population with progressively decreasing ‘Asian’ components in the genome from east to west in Europe. This type of information would shed light on the expansion history of the parasite in eastern Europe, which may have been colonized by E. multilocularis populations from both the west (Alpine focus) and the east (Asia).

This study was initially designed in order to confirm the hypothesis that the E. multilocularis presence in eastern European countries resulted from an expansion from the Alpine historical focus ruled by the mainland-island system of transmission previously described. The secondary objectives for a better understanding of the expansion history of the parasite were: (i) to propose a spatial scenario of E. multilocularis expansion throughout Europe; (ii) to investigate the potential overlapping of European and Asian clades of E. multilocularis in Eurasia.

Section snippets

Sample collection and EmsB genotyping

The E. multilocularis samples were mainly collected as part of previous studies in each country or through convenience sampling (Table 1, Fig. 1). Worms were mainly collected from the intestines of red foxes, but also from raccoon dogs and golden jackals. A maximum of five worms per definitive host were analyzed (except for six worms from one of the two foxes from Ukraine and from one of the four foxes from Turkey). Larval stages (metacestodes) were obtained from humans, a captive Senegal

Western Europe

The 176 samples from Germany, Belgium, Luxembourg and the Netherlands were grouped into 14 EmsB profiles named WE1 to WE14 (Fig. 2). According to the accumulation curves, the sampling was representative of all EmsB profiles for Luxembourg and South Germany and to a lesser extent for northern Belgium (Fig. 3A), as also indicated by the species richness estimation of the total number of profiles from these areas (Table 2). The genetic diversity index was low for Germany but moderate for northern

Discussion

The relatively high number of sampled E. multilocularis isolates in this study were from a large number of countries, including nine eastern European countries. In some cases, due to the convenience sampling approach, sampling did not cover the countries as a whole, and may reflect a regional scale rather than a country scale. Nevertheless, this is the first EmsB genotyping study for most of these eastern European and Asian countries, where even mitochondrial sequencing studies are scarce. The

Conclusion

In Europe, a mainland-island system of transmission of E. multilocularis appears to have resulted in expansion from the historical Alpine focus to the peripheral areas of Luxembourg, Belgium and the Netherlands. The presence of the parasite in eastern European countries also resulted from gradual eastward expansion from the historical focus. Despite the previous identification of an Asian admixture in European E. multilocularis populations in Poland, no EmsB profiles from the Asian clade were

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors wish to thank Titia Kortbeek (Centre for Infectious Diseases Research, Diagnostics and Laboratory Surveillance, National Institute for Public Health and the Environment) for providing alveolar echinococcosis human samples from the Netherlands. This study was partially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Contract No. 451-03-68/2020-14/200007 and Contract No. TR31084.

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