Rapport 11- 2019

Status of Echinococcus multilocularis in Svalbard - Final Report to Svalbard Environmental protection Fund NORWEGIAN VETERINARY INTSTITUTE

Status of Echinococcus multilocularis in Svalbard Preface

This is the final report of the project “Status of Echinococcus multilocularis in Svalbard” 16/42. Funding for this project was allocated by the Svalbard Environmental Protection Foundation in the spring of 2016. The project was carried out by a team of researchers from the Norwegian Veterinary Institute and the Norwegian Polar Institute, who for the first time collaborated in this project. The Norwegian Veterinary Institute has contributed with expert knowledge on E. multilocularis, pathology, parasitological and molecular methods, and the Norwegian Polar Institute with specific expertise on Arctic foxes, sibling voles, E. multilocularis and environmental aspects. In addition, we have benefitted from working together with Fredrik Samuelsson, Svalbard guide with an MSc in Parasitology, whose local knowledge has greatly facilitated the sample collection.

The results of our project were presented to locals at a seminar at the University Centre in Svalbard 19.11.2018.

We gratefully acknowledge the Svalbard Environmental Protection Fund for supporting our project. We would also like to thank Paul Lutnæs/the Govenor of Svalbard, Rupert Krapp from the Norwegian Polar Institute and the Svalbard Vets for assisting in collecting samples for the project; Longyearbyens Hundeklubb, commercial dog sledging companies and private dog owners who kindly let us collect faecal samples from their dogs; arctic fox hunters who donated foxes for our study, and inhabitants in Longyearbyen who helped trapping sibling voles.

In this project we have collaborated with Dr. Jenny Knapp from University of Bourgogne Franche-Comté, France to characterise the E. multilocularis isolates using microsatellite markers. This work was funded separately. The combined research activities have been presented in the scientific publication: Enemark HL, Woolsey ID, Fuglei E, Knapp J, Madslien K, Samuelsson F, Mørk T, Øines Ø, 2019. Status of Echinococcus multilocularis on the archipelago of Svalbard two decades after the first detection of the parasite. International Journal for Parasitology: Parasites and Wildlife XXXXX

Oslo, 12 April 2019

Heidi L. Enemark Project Leader

Collection of faecal samples from sledge dogs. Photo: Heidi L. Enemark

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Contents

Summary

1. Introduction 6 1.1. Background 6 1.2. Objectives 7 2. Materials and methods 8 2.1. Sample collection 8 2.1.1. Sibling voles 8 2.1.2. Arctic foxes 10 2.1.3. Faeces from domestic dogs 10 2.2. Laboratory analyses 11 3. Results and discussion 11 3.1. Sibling voles 11 3.2. Arctic foxes 12 3.3. Domestic dogs 13 3.4. Concluding remarks 14 4. References 15 Appendix a) Project information to dog owners b) Questionnaire

Authors: ISSN 1890-3290 © Veterinærinstituttet 2019 Heidi L Enemark Eva Fuglei Funding: Ian D. Woolsey Svalbard Environmental Protection Fund Fredrik Samuelsson Torill Mørk Kristin Henriksen Knut Madslien Øivind Øines Photo front cover: Heidi L. Enemark

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Contact information:

Heidi L. Enemark, [email protected] Ian D. Woolsey, [email protected] Kristin Henriksen, [email protected] Knut Madslien, [email protected] Øivind Øines, [email protected]

Norwegian Veterinary Institute Ullevålsveien 68 Pb 750 Sentrum NO-0106 Oslo Norway

Torill Mørk, [email protected]

Norwegian Veterinary Institute Stakkevollveien 23 NO-9010 Tromsø Norway

Eva Fuglei, [email protected]

Norwegian Polar Institute Fram Centre NO-9296 Tromsø Norway

Fredrik Samuelsson [email protected] Independent consultant

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Summary

In this project we studied the prevalence of the zoonotic tapeworm Echinococcus multilocularis in final hosts, arctic foxes (Vulpes lagopus) and domestic dogs (Canis lupus familiaris), as well as in intermediate hosts, sibling voles (Microtus levis), in Svalbard, Norway. The overall aims were to reveal potential public health risks associated with the presence of E. multilocularis in Svalbard and uncover possible needs for interventions. In addition, presence of other gastro-intestinal parasites was analysed in dogs.

E.multilocularis was recently ranked as the most import foodborne parasite in Europe due to its public health importance. The definitive hosts are infected through prey of infected rodents and shed eggs in their faeces without any signs of disease. Humans may become accidental intermediate hosts by ingestion of contaminated food/water, via environmental contamination or close contact with infected dogs. If left untreated, the mortality is close to 100% in humans.

The samples (sibling voles (n=29) and faeces from dogs (n=89) and arctic foxes (n=304) were mainly collected between November 2014 and May 2017, but 38 “historical” samples from foxes collected between 2009 and 2013 were also included. To analyse for presence of E. multilocularis in the faecal samples, we used a highly sensitive molecular method (DNA fishing combined with real-time PCR detection), which is also used in the surveillance of E. multilocularis in mainland Norway to prove freedom of disease. This method can detect as little as one egg in 3 g of faeces, but it only tests positive if the faecal samples are collected during the patent phase of the infection i.e. when adult tapeworms are present in the intestines of infected final hosts. Faecal samples from dogs were also examined for presence of other gastrointestinal parasites using the McMaster method, and information about age, breed, housing, travel activity, deworming practices and health was collected for each dog in a questionnaire. With the assistance from the local inhabitants, sibling voles were trapped in and around Longyearbyen. These voles were later forwarded to the Veterinary Institute in Tromsø, where they were autopsied to establish possible liver changes caused by E. multilocularis.

No E. multilocularis positive sibling voles were detected in our study. However, due to an insufficient sampling scheme many of the voles were decomposed or dried out and therefore not suited for autopsy. Consequently, we cannot rule out that we might have missed positive cases. Nevertheless, sibling voles were found all over Longyearbyen, and considering the ongoing climate changes they may spread/propagate even further.

The overall prevalence of E. multilocularis in arctic foxes, 2014-2017, was 7.9%. In accordance with previous findings in Svalbard, all positive cases with one exception were detected within a radius of 60 km from Grumant, the core area of the sibling voles/E. multilocularis. A single E. multilocularis positive arctic fox was detected in the town of Longyearbyen, and even though no positive sibling voles were found, the theoretical conditions for the establishment of the E. multilocularis lifecycle may be present depending on the population densities of the hosts/intermediate hosts. An additional positive arctic fox was found on the island of Hopen, approximately 300 km from the core area of Grumant. These findings are worrying because they suggest that the infection may be transmitted to humans not only in the Grumant area, but also in other parts of Svalbard. The risk of infection outside the Grumant area is probably low though, as previous studies have clearly demonstrated that the risk of infection in foxes decreases with distance from Grumant.

No dogs in the present investigation tested positive for E. multilocularis and low grade infections of other gastrointestinal parasites were only found in two dogs. Thus we could not perform a proper risk assessment. The results may indicate that the current deworming practices in Longyearbyen are sufficient. However, these results are based on a small sample size, and we therefore suggest that future studies should include a larger number of dogs, represent all age groups and preferably be based on serological detection of antibodies against E. multilocularis. Finally, surveillance of E. multilocularis in arctic foxes as well as in sibling voles is warranted to monitor further geographical spread of the infection in Svalbard.

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1. Introduction 1.1. Background

Climate changes in the Arctic, result in rapid environmental and ecosystems changes (Post et al., 2009; Gilg et al., 2012), which in turn are expected to affect parasite transmission (Davidson et al., 2011). As a key predator in a simple and well-investigated terrestrial predator-prey-system (Ehrich et al., 2015), the arctic fox (Vulpes lagopus) is one of ten flagship species selected by the International Union for Conservation of Nature to represent and monitor consequences of climate changes on the tundra ecosystem (IUCN, 2009). Since changes in dynamics of parasitism in the Arctic may have direct influence on wildlife populations (Davidson et al., 2011) as well as humans, baseline knowledge on parasite diversity in this key predator can increase our knowledge on parasite-host-relationships, and thereby contribute to identify effects of climate changes on population dynamics in Svalbard. Ideally, this should be monitored regularly throughout several decades.

The zoonotic (i.e. transmissible between animals and humans) and highly pathogenic tapeworm E. multilocularis was first detected in Svalbard in 1999 (Henttonen et al., 2001). Definitive hosts of this parasite are canids including e.g. arctic foxes and domestic dogs (Canis lupus familiaris) while rodents, in Svalbard the sibling voles (Microtus levis), are intermediate hosts. The definitive hosts are infected through prey of infected rodents and shed eggs in their faeces without any signs of disease. The eggs are resistant to low temperatures and can remain infective for several months (Fig. 1). Humans may become accidental intermediate hosts by ingestion of contaminated food/water, via environmental contamination or close contact with infected dogs (Nagy et al., 2011). Following an asymptomatic incubation period of several years (5-15 years) (WHO, 2014) alveolar echinococcosis develops; one of the most severe zoonotic infections in the northern hemisphere. Infections in humans are rare but cause considerable public health concern due to treatment costs and high mortality if left untreated (Torgerson et al., 2008). Based on public health importance, E. multilocularis was ranked third by importance globally relative to other foodborne zoonotic parasites (FAO/WHO, 2012). However, a recent risk ranking conducted by a group of European experts, ranked E. multilocularis as the most important foodborne parasite in Europe (Bouwknegt et al., 2018).

Previous studies in Svalbard have shown that high prevalence of E. multilocularis in foxes was directly related to the local vole abundance i.e. to the high density of sibling voles found in the Grumant area (Fuglei et al., 2008; Stien et al., 2010). Variations in vole abundance are highly dependent on icing of the tundra due to “rain-on-snow” events (Stien et al., 2012; Hansen et al., 2013). In years with high population density, the voles are more likely to spread to Longyearbyen. Through a recent project financed by the Svalbard Environmental Protection Fund we know that arctic foxes move on average 6.6 km per day (from 0.5 to 72.6 km), which means that foxes from the core-area of the sibling vole, i.e. Grumant, may easily reach Longyearbyen (Fuglei et al., 2016). When this project was initiated, there was no knowledge about the local sibling vole population in Longyearbyen or the E. multilocularis infection rate of these animals, and therefore our project intended to fill the knowledge gap.

Growing numbers of sibling voles are likely to increase the risk of echinococcosis in dogs. In particular sledge dogs kept outdoors may probably be at risk of infection due to easier access to consumption of sibling voles. Thus, they could represent a particular public health risk for both residents and tourists on the island. Higher prevalence of echinococcosis in arctic foxes coming into Longyearbyen will also increase the public health risks via increased environmental contamination with E. multilocularis eggs. Before this study was initiated, nothing was known about the role of dogs in the lifecycle of E. multilocularis in Svalbard, but studies have shown contact with dogs to be a risk factor for alveolar echinococcosis in humans, and infection of humans through the transmission of E. multilocularis eggs after direct contact with dogs is conceivable (Nagy et al., 2011). Therefore, we wanted to conduct a prevalence study and a risk factor analysis of E. multilocularis and other parasites of dogs (e.g. Taenia spp., Toxascaris leonina, and Strongyloides stercoralis, which have been found e.g. in arctic foxes from Greenland (Andreassen et al., 2017)), and carry out prevalence studies in arctic foxes as well as sibling voles trapped in and around

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Longyearbyen. Such data can be used to identify possible risks for humans and needs for adjustment of management and deworming practices of dogs.

Finally, genetic analyses of E. multilocularis isolates collected from sibling voles in the Grumant area during 2004-2006 demonstrated that the current infection most likely was introduced to the island by roaming arctic foxes and sustained by the local population of sibling voles introduced by early settlers (Knapp et al., 2012). The isolated ecosystem of Svalbard constitutes an ideal area for studying genetic drift, i.e. change in the frequency of a gene variant (allele) in a population of E. multilocularis, and this project has therefore collaborate with Laboratory of Chrono-environnement, France to illuminate the genetic changes that have taken place in the worm population throughout the past decade.

A B Fig. 1 Adult E. multilocularis (A) isolated from the intestine of an infected fox. The adult stage of the tapeworm is 2-4 mm long. In the sack-like uterus, the eggs (up to 200) are clearly visible. The eggs (magnified in image B) can survive for several months in the environment. When they are ingested by an intermediate host (rodent such as e.g. a sibling vole) or an accidental host (human) multiple cysts are formed in the liver causing alveolar echinococcosis, which is highly pathogenic. Photos: Heidi L. Enemark

1.2. Objectives

The overall aims of this project were to reveal potential public health risks associated with the presence of E. multilocularis in Svalbard and uncover possible needs for interventions.

The specific aims were to:

 Determine the prevalence of E. multilocularis in the intermediate host (sibling voles) in Longyearbyen, Svalbard.

 Establish prevalence of E. multilocularis in the final hosts (arctic foxes and dogs), and risk factors for echinococcosis in dogs.

 Uncover the prevalence of gastrointestinal parasites other than E. multilocularis in dogs.

An additional aim was to determine the genetic profiles of selected E. multilocularis isolates and compare these with previously published isolates collected in Svalbard to describe genetic changes over time and study if multiple introductions of the parasite to Svalbard may have occurred. This was done in collaboration with Dr. Jenny Knapp, Department of Chrono-environnement, University of Bourgogne Franche-Comté, France. These molecular analyses were not financed by the Svalbard Environmental Protection Fund and have been reported separately in a scientific publication (Enemark et al., XXXXX)

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2. Materials and methods 2.1. Sample collection

The overall study period was from the 1st of June 2016 to 31st of December 2018. However, during this period we decided to increase the number of faecal samples from arctic foxes by adding samples collected during previous seasons as described below. 2.1.1. Sibling voles

In October 2016 an article about our project was published in Svalbardposten to create awareness about E. multilocularis and to invite inhabitants in Longyearbyen to assist trapping sibling voles for the project. In November 2016, a total of 100 metal mouse traps were handed out to people who had expressed interest in the project and the traps were distributed in and around Longyearbyen (at the Islandic horse stable near the airport, at the veterinary clinic and guest houses in Nybyen, at the “Longyearbyens Hundeklubb” and at Green Dog Husky farm). The traps were checked at irregular intervals, and the voles were delivered to Rupert Krapp at the Norwegian Polar Institute at Forskningsparken in Longyearbyen. Additional traps (n=30) were distributed in May 2017. A total of 29 sibling voles were sampled from 7 different locations (Table 1) and stored at -20O C until shipment to the Veterinary Institute in Tromsø.

Fig 2. Nybyen, Longyearbyen, Svalbard. Example of a location where several sibling voles were trapped in the basements of the houses.

Fig 3. Horse stable near the airport approximately, 3 km from the town centre of Longyearbyen, Svalbard. An example of a location where sibling voles were trapped.

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Table 1. Date of collection, location, body weight/length and sex of sibling voles (Microtus levis) examined for presence of Echinococcus multilocularis.

ID Date Location Weight Length Sex Histology Comments (g) (cm) 2018/80/23.1 06.11.2016 Horse stable 21,4 10,3 Female Yes 2a 03.05.2017 Horse stable 7,5 Unknown Desiccated 2b 03.05.2017 Horse stable 7,6 Unknown Desiccated 3 11.11.2016 17,9 9,5 Yes 4 04.05.2017 Longyearbyen 31,7 11,2 Male Yes dog club 5 07.11.2016 Guesthouse 33,7 11,6 Female Yes Suspicious 102 Nybyen liver changes AE not verified by histology 6 20.12.2016 Guesthouse 21,6 11,1 Female Yes 102 Nybyen 7 03.05.2017 Guesthouse 16 9,7 Yes Decomposed 102 Nybyen 8 16.11.2016 Stormessen 23,3 10,3 Female Yes Nybyen 9 09.11.2016 Stormessen 27,8 11,8 Female Yes Nybyen 10a Desiccated 10b 11 16.11.2016 Stormessen 38 12,1 Male Yes Nybyen 12 07.11.2016 16,1 8,9 Male Desiccated 13a 24.11.2016 Green Dog 27,2 11 Male Yes Desiccated 13b 24.11.2016 Green Dog 14,9 7,9 Male 13c 24.11.2016 Green Dog 13d 24.11.2016 Green Dog 13e 24.11.2016 Green Dog 14a 04.11.2016 Horse stable 31,9 11,3 14b 04.11.2016 Horse stable 22 9,6 14c 04.02.2016 Horse stable 14d 04.02.2016 Horse stable 15a 15b 15c 16a 27.11.2016 Longyearbyen hundeklubb 16b 27.11.2016 Longyearbyen hundeklubb 16c 27.11.2016 Longyearbyen hundeklubb

Blanks: No information provided/could not be determined.

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2.1.2. Arctic foxes

Faecal samples were collected by the Norwegian Polar Institute from trapped arctic foxes and from arctic foxes found dead of natural causes. In addition, a few foxes were shot and provided by fox hunters. Samples from trapped arctic foxes were collected on Spitsbergen, Svalbard, mainly around the Isfjorden area (77.8-79.1oN, 13-17oE), and a few were also collected around Austfjordnes (79.12oN, 16.18oE). Baited traps were used to collect the foxes during the annual harvest between the 1st of November and 15th of March in 2009/2010 and 2012/2012 (n=38), 2014/2015 (n=154), 2015/2016 (n=32) and 2016/2017 (n=72). Samples from arctic foxes that were found dead of natural causes were collected from the nearby areas of Longyearbyen (2013 n=1, 2014 n=1, 2015 n=3, 2016 n=1), and from Bjørnøya (74.44oN, 19.02oE; 2015 n=1). All foxes were frozen at -80o C for 7 days before they were weighted, sex-determined, and skinned prior to the final autopsies were conducted using the laboratory facilities at the Veterinary Institute in Tromsø. The age of the arctic foxes was determined by counting the annuli in the cementum of a sectioned canine tooth (Grue and Jensen 1976). A total of 304 faecal samples from artic foxes were included in our molecular analyses for presence of E. multilocularis. All samples were stored at -20o C until analysis.

2.1.3. Faeces from domestic dogs

Invitations to participate in the study were emailed to Longyearbyen hundeklubb, where most privately owned dogs in Svalbard are housed when they are not used for sledging. In addition, invitations (Appendix A) were posted at various locations in Longyearbyen e.g. at the supermarked, Svalbardbutikken, and at the veterinary clinic in Nybyen. Only dogs that had not been dewormed within a period of minimum 3 months were included in the study. Individual faecal samples were collected 3-6 October 2016 (n=17) and 2-4 May 2017 (n=72) from a total of 89 dogs. Samples (>50g) from 25 privately owned dogs were collected by the owners. These samples were delivered to Svalbard Vets where they were stored at approximately 5o C until they were brought to the Veterinary Institute in Oslo for laboratory analyses. The remaining samples (n=64) were collected by Fredrik Samuelsson and researchers from the Veterinary Institute from sledge dogs from commercial dog farms and from Longyearbyen Hundeklubb (Fig. 4). For each dog a short questionnaire (Appendix B) was filled in by the owner or by the researchers after having interviewed the owners. Questions that could be related to increased risk of echinococcosis such as age, breed, housing, travel activity, deworming practices and health were included in the form.

Fig 4. Collection of faecal samples from sledge dogs from commercial dog farms. The samples were subjected to general parasitological analysis and specific molecular analysis for the presence of Echinococcus multilocularis. Photo: Heidi L. Enemark

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2.2 Laboratory analyses

Autopsy of sibling voles: Necropsy was conducted in February 2018, and weight, length and sex were registered when possible.The voles were examined macroscopically with specific focus on liver abnormalities/alveolar echinococcosis. If pathological liver or lung changes were observed supplementary standard histological analyses were performed to examine for specific parasitic structures.

Molecular analyses for presence of E. multilocularis: 3 g faecal samples from both arctic foxes (n=304) and dogs (n=89) were analysed by a highly sensitive method (DNA-fishing combined with real-time PCR detection). This involved magnetic capture DNA extraction from the samples by applying specific DNA- hybridisation, followed by extraction using streptavidin coated magnetic beads and final detection by real-time PCR (Øines et al., 2014; Isaksson et al., 2014). This approach enables detection of E. multilocularis DNA from eggs as well as adult worms during the patent phase of the infection when eggs are shed in the faeces. The method is capable of detecting approximately 1 egg per 3 g of faeces.

General parasitological analysis: 4 g faecal samples from all dogs (n=89) were analysed for presence of gastro-intestinal parasites using a modified centrifugation-enhanced McMaster method (Henriksen et al., 1976). By this method, parasite eggs/oocysts are quantified and identified by microscopy following flotation using a solution with high density. The method has a sensitivity of 5 eggs per g of faeces. Taeniid eggs including those of E. multilocularis can be detected by this method, but molecular methods (PCR) are required to differentiate the eggs from eggs excreted by other tapeworms.

A B

Fig. 5 Reception and storage of faecal samples from dogs at the veterinary clinic (Svalbard Vets) in Longyearbyen (A). Packing of the samples before transport to mainland Norway and laboratory analysis at the Veterinary Institute in Oslo. Photo: Heidi L. Enemark (A); Fredrik Samuelsson (B). 3. Results and discussion 3.1. Sibling voles

None of the 29 sibling voles examined by necropsy had visible pathological changes corresponding to alveolar echinococcosis. However, a large proportion (31%) of the voles were desiccated or decomposed, and therefore pathological changes might have been missed. Thus, the negative outcome of the analyses should be evaluated with caution. Taking into consideration that the prevalence in sibling voles was among the highest ever recorded (close to 100% in overwintered males) when the parasite was first detected in 1999 (Henttonen et al., 2001), it seems possible that sibling voles in Longyearbyen less than 20 km from Grumant, may also be at risk of infection, particularly considering the fact that this study found an E. multilouclaris infected arctic fox in Longyearbyen (see 3.2).

Although we did not detect any E. multilocularis positive sibling voles in Longyearbyen, our study verified that the sibling voles have spread geographically from the Grumant area to Longyearbyen, and therefore the lifecycle of E. multilocularis can theoretically be sustained, although the likelihood of transmission will depend on the population density of both intermediate and final hosts.

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To increase the quality of the analyses, future studies should improve the sampling scheme so that mouse traps are emptied regularly and before the dead voles reach a state where they are unfitted for autopsy. Additionally, the macroscopic and microscopic examination of liver changes should be supplemented with molecular analyses using E. multilocularis specific genetic markers to increase the sensitivity of the analyses.

3.2. Arctic foxes

Of the 304 fox samples analysed in the present study, 17 were positive for E. multilocularis (Table 2). This corresponds to an overall prevalence of 5.6% in the foxes. However, none of the samples collected before the trapping season 2014/2015 were positive for E. multilocularis and we therefor think that the parasite DNA in these samples may have been of poor quality or destroyed due to the long-term storage. Our results may also have been biased by the relatively large proportion of samples (approximately 1/3) with less than 3 g faeces, which is required for an optimal/sensitive test outcome.

Assessment of the prevalence of E. multilocularis based only on samples collected from 2014 to 2017 resulted in a prevalence of 7.9%. This is in accordance with previous findings of prevalences in arctic foxes trapped at some distance from the core area of the sibling voles and where the E. multilocularis is detected in the voles, namely in Grumant. Higher prevalences have been documented by Fuglei et al. (2008), however, only in foxes in Grumant.

Table 2. Geographical origin, year of collection, sex, body weight and age of Echinococcus multilocularis positive Arctic foxes (Vulpes lagopus), 2014 to 2017.

Sample ID Location Year Sex Body weight Age kg tooth 2014-15 2 Gangdalen NA F 2.7 7 2014-15 10 Diabasodden 2015 M 3.5 2 2014-15 19 Sveagruva 2015 M 3.8 3 2014-15 36 Blåhuken 2015 F 2.5 2 2014-15 47 Colesbukta 2015 F 3.1 1 2014-15 48 Colesbukta 2015 F 3.7 1 2014-15 57 De Geerdalen Sør 2015 M 3.0 1 2014-15 59 Foxdalen 2015 M 3.1 1 2014-15 71 Istjørndalen 2015 M 3.2 2 2014-15 78 Skardalen 2015 F 2.5 1 2014-15 121 Kapp Schollin 2015 M 3.6 2 2014-15 126 Kolfjellet 2015 F 3.4 1 2015-16 18 Bellsund 2016 M 2.8 5 2016-04-TØ102 Longyearbyen 2013 F 3.3 3 2016-04-TØ105 Krykkjefjellet, Hopen 2015 M 2.7 2 2016-04-TØ116 Revneset 2016 M 2.4 2 2016-2017 40 Colesbukta 2017 M 4.7 1

One E. multilocularis positive arctic fox, dead of natural causes, was detected in Longyearbyen, and all of the positive foxes, apart from one, were detected south of Isfjorden within a radius of 60 km from the Grumant area. This result was anticipated, as a previous study has demonstrated a close correlation between the prevalence of E. multilocularis in foxes and distance to the Grumant area. In 2000, when that study was conducted, 7 of 37 foxes from Grumant tested positive for E. multilocularis by coprogen ELISA (Fuglei et al., 2008). However, it is important to note that these results are not directly comparable

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as coproantigen ELISA may over interpret the prevalence due to false positive results, and therefore prevalence estimates based on coproantigen ELISAs are expected to be higher than prevalences based of PCR analyses of faecal samples.

One positive fox, dead of natural causes, was detected on the island of Hopen approximately 300 km away from Grumant. This is in accordance with earlier findings that positive foxes may be detected far away from the core area of E. multilocularis/sibling voles (Stien et al 2010, more than 70 km from Grumant), simply because the arctic fox is very mobile. The arctic fox from Hopen that tested positive may also have brought the parasite from other places in the Arctic where the parasite is known to be present.

Thus, although E. multilocularis positive foxes are mainly found in the Grumant area, our results demonstrate that that infective eggs of E. multilocularis may be detected in larger parts of Svalbard despite the facts that so far the intermediate host has only been found within a relatively narrow radius from Grumant where is was first introduced. Nevertheless, close monitoring of the population of sibling voles in Svalbard is necessary to estimate the risk of further geographical spread of the parasite. Likewise, regular monitoring of the prevalence of E. multilocularis in arctic foxes is warranted to estimate the public health risks in Svalbard.

Fig. 6. Geographical origin of the arctic foxes positive for E. multilocularis. With one exception, all positive foxes were found within a radius of 60 km from the Grumant area, where E. multilocularis was first detected in 1999 (Henttonen et al., 2001).

3.3. Domestic dogs

Faecal samples for parasitological analysis was provided by the owners of 25 dogs whereas the remaining 64 samples were collected mainly from the commercial dog farms around Longyearbyen. All samples tested negative by molecular analysis/PCR for E. multilocularis, and therefore we were unable to perform a risk analysis that could identify risk factors for infection with E. multilocularis in dogs. We would have liked to include more dogs in our studies, but it was a challenge to find dogs that had not been dewormed shortly before sampling. All dogs apart from one were ≥1 year. Adult age should increase the risk of infection with E. multilocularis (because the dogs have to ingest infected sibling voles to become infected), whereas young age increases the risk of infection with other gastrointestinal parasites. Thus, it was no surprise that gastrointestinal parasites were only found in two dogs: coccidia (Cystoisospora spp.

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6400 oocysts per g of faeces) in one dog, and roundworms (Toxascaris leonina 10 eggs per g of faeces) in another dog. None of these dogs had clinical symptoms.

Four dogs were registered as pets by the owners, whereas all other dogs were sledge dogs. One English setter was included while the other dogs in the study were Huskies (Alaskan, Siberian, Greenlandic or mixtures of these breeds). Of the 89 dogs 18 had been travelling to Europe or the United States. Two dogs were dewormed less than twice yearly, 9 were dewormed more than two times per year and the vast majority (87.6%) was dewormed regularly at 6 months intervals.

3.4. Concluding remarks

Our study verified the presence of E. multilocularis in the arctic fox population of Svalbard. For the first time a positive fox was found in Longyearbyen. Another positive fox was detected on the island of Hopen, approximately 300 km from the core area of Grumant. In accordance with earlier studies, our findings prove that people may be at risk of infection even in areas far from Grumant. However, as demonstrated previously, E. multilocularis was primarily detected in foxes in the vicinity of the Grumant area. The overall prevalence found in our study was relatively low (7.9%), but in agreement with previous findings.

The prevalence of E. multilocularis in red foxes is know to vary throughout the year and between seasons depending on climate, availability of food, density of the population of intermediate hosts etc. Similarly, in the Grumant area the population of sibling voles varies from year to year depending e.g. on climate conditions, and that there is a correlation between the density of the vole population and presence of E. multilocularis in the arctic foxes (Fuglei et al., 2008). The transmission dynamics of E. multilocularis outside of Grumant still remain to be elucidated.

No E. multilocularis positive sibling voles were detected in our study. Nevertheless, a total of 29 sibling voles were trapped in Longyearbyen or within a short distance from the town center. Due to an insufficient sampling scheme (with did not have the capacity in this project to empty the traps on a daily basis) many of the voles were decomposed or dried out and therefore not suited for autopsy. Therefore, we cannot rule out that we might have missed a positive case.

In this study, we have documented the presence of sibling voles all over Longyearbyen, and considering the ongoing climate changes they may spread even further. We also detected a positive arctic fox in the town and even though no positive sibling voles were detected, the theoretical conditions for establishment of the E. multilocularis lifecycle seem to be present i.e. final host and intermediate hosts sharing the same environment.

We did not detect E. multilocularis in any dogs in the present study. Considering the close contact between dogs and humans (owners as well as tourists) this was a reassuring finding. However, our results should be interpreted with caution as the sample size was too small to make any firm conclusions regarding the prevalence of E. multilocularis in dogs in Svalbard. Other studies have shown that the prevalence of E. multilocularis in final hosts varies throughout the year depending on several factors. Therefore, future studies should include more dogs, different age groups and samples taken at diverse time points to reflect possible seasonal variation in the prevalence. Preferably, serological methods should be used as they allow detection of E. multilocularis antibodies even if the infection has been cleared.

Based on previous studies and our current findings, we recommend that:  Every effort should be taken to reduce the population of sibling voles in Longyearbyen.  Use of the Grumant area for recreational and tourist purposes should be minimized.  Fox hunters exercise caution when handling foxes. Preferably, they should wear disposable gloves and surgical masks, particularly if the foxes are shot in the Grumant area.  Dogs are dewormed regularly. Only deworming at monthly intervals will secure freedom of infection, if no monitoring is in place.

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 Dogs are prevented from eating rodents.  Sand pits are covered to avoid faecal contamination from infected foxes.  Proper hygiene is enforced. Frequent hand washing, particularly after handling potential final hosts (arctic foxes and dogs) and before meals, will prevent infection.  Water from natural sources should be heat treated before consumption.  Regular information about this zoonotic infection should be given to local inhabitants and visitors to Longyearbyen

Finally, surveillance of E. multilocularis in arctic foxes as well as in sibling voles is warranted to monitor further geographical spread of the infection in Svalbard.

4. References

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Knapp J, Staebler S, Bart JM, Stien A, Yoccoz NG, Drögemüller C, Gottstein B, Deplazes P, 2012. Echinocuccus multilocularis in Svalbard, Norway: microsatellite genotyping to investigate the origin of a highly focal contamination. Infection, Genetics and Evolution 12: 1270-1274. Madslien K, Albin-Amiot C, Jonsson ME, Clausen T, Henriksen K, Urdahl AM, Heier BT, Øines Ø. The surveillance programme for Echinococcus multilocularis in red foxes (Vulpes vulpes) in Norway in 2014. Surveillance programmes for terrestrial and aquatic animals in Norway. Annual report 2014. Oslo: Norwegian Veterinary Institute 2015. Nagy A, Ziadinoy L, Schweiger A, Schnyder M, Deplazes P, 2011. [Hair coat contamination with zoonotic helminth eggs of farm and pet dogs and foxes]. Berl Munch Tierarztl Wochenschr 124: 503-511. Post E, Forchhammer MC, Bret-Harte MS, Callaghan TV, Christensen TR, Elberling B, Fox AD, Gilg O, Hik DS, Høye TT et al. 2009. Ecological Dynamics Across the Arctic Associated with Recent Climate Change. Science 325: 1355-1358. Stien A, Voutilainen L, Haukisalmi V, Fuglei E, Mørk T, Yoccoz NG, Ims RA, Henttonen H, 2010. Intestinal parasites of the arctic fox in relation to the abundance and distribution of intermediate hosts. Parasitology 137: 149-157. Stien A, Ims RA, Albon SD, Fuglei E, Irvine RJ, Ropstad E, Halvorsen O, Langvatn R, Loe LE, Veibarg V, Yoccoz NG, 2012. Congruent responses to weather variability in high-arctic herbivores. Biology Letters 8: 1002-1005. Torgerson PR, Schweiger A, Deplazes P, Pohar M, Reichen J, Ammann RW et al., 2008. Alveolar echinococcosis: from a deadly disease to a well-controlled infection. Relative survival and economic analysis in Switzerland over the last 35 years. Journal of Hepatology 49: 72-77. WHO, 2014. Report of the WHO Informal Working Group on Echinococcosis on the occasion of the XXV World Congress of Echinococcosis held in Khartoum, Sudan, 25 November 2013. Available online: http://www.who.int/echinococcosis/WHO_HTM_NTD_NZD_2014.1/en/ Øines O, Isaksson M, Hagstrom A, Tavornpanich S, Davidson RK, 2014. Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces. Parasites & Vectors 7: 246

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Appendix

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Final Report to Svalbard Environmental Protection Fund

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