Assessment of Echinococcus multilocularis surveillance reports submitted 2013 in the context of Commission Regulation (EU) No 1152/2011 1

EFSA Journal 2013;11(11):3465 SCIENTIFIC REPORT OF EFSA Assessment of Echinococcus multilocularis surveillance reports submitted 2013 in the context of Commission Regulation (EU) No 1152/2011 1 European Food Safety Authority 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT This report provides an analysis and critical assessment of the sampling strategy, the data collected, and the detection methods used in the E. multilocularis surveillance carried out in Finland, Ireland, Malta and the UK and reported in 2013 in the context of Regulation (EU) No 1152/2011 regarding preventive health measures for the control of E. multilocularis infection in dogs. The surveillance aims at detecting the parasite, if present in any part of those Member States. The 2013 surveillance reports of the four Member States were assessed by checking the description of the surveillance system for completeness against the relevant elements that need to be addressed in assessing the quality of E. multilocularis surveillance reports in the context of Regulation (EU) No 1152/2011. The data reported on individual samples were assessed using the raw data submitted by the Member States via the EFSA Data Collection Framework (DCF). Descriptive statistics were calculated to check whether the requirements of Regulation (EU) No 1152/2011 had been fulfilled. None of the four Member States who are operating an E. multilocularis-specific surveillance programme aimed at detecting the parasite, if present in any part of those Member States, has detected E. multilocularis through the surveillance activities reported in 2013. Under the assumption of unbiased representative sampling (in the case of Finland, Ireland and the UK) and unbiased risk based sampling (in the case of Malta) and considering the sensitivity of the tests applied, all four MS have fulfilled the requirement of Regulation (EU) No 1152/2011 to the effect that the surveillance activities should detect a prevalence of E. multilocularis of 1 % or less at a confidence level of at least 0.95. European Food Safety Authority, 2013 KEY WORDS Echinococcus multilocularis, absence of infection, freedom from disease, surveillance 1 On request from the European Commission, Question No EFSA-Q-2013-00535, approved on 12 June 2012. 2 Correspondence: AHAW@efsa.europa.eu 3 Acknowledgement: EFSA wishes to thank the members of the EFSA Scientific Network for Risk Assessment in Animal Health and Welfare: Antti Oksanen, Fabrizio Rosso, Helen Roberts, June Fanning, Marja Isomursu, William Byrne, and the EFSA Animal Health and Welfare Panel members: Mariano Domingo and Preben Willeberg for the preparatory work on this scientific output, and EFSA staff: Andrea Gervelmeyer, Gabriele Zancanaro, Jane Richardson and Andrea Baù for the support provided to this scientific output. Suggested citation: European Food Safety Authority, 2013. Assessment of Echinococcus multilocularis surveillance reports submitted 2013 in the context of Commission Regulation (EU) No 1152/2011. EFSA Journal 2013;11(11):3465, 41 pp. doi:10.2903/j.efsa.2013.3465 Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2013

SUMMARY Following a request from the European Commission, the EFSA Animal Health and Welfare Unit (AHAW) was asked to provide scientific and technical assistance on E. multilocularis infection in animals. This report provides an analysis and critical assessment of the sampling strategy, the data collected, and the detection methods used in the E. multilocularis surveillance carried out in Finland, Ireland, Malta and the UK and reported in 2013 in the context of Regulation (EU) No 1152/2011 regarding preventive health measures for the control of E. multilocularis infection in dogs. The surveillance aims at detecting the parasite, if present in any part of those Member States. The 2013 surveillance reports of the four Member States were assessed by checking the description of the surveillance system for completeness against the relevant elements that need to be addressed in assessing the quality of E. multilocularis surveillance reports in the context of Regulation (EU) No 1152/2011. In a second step, the data reported on individual samples were assessed using the raw data submitted by the Member States via the EFSA Data Collection Framework (DCF) and descriptive statistics were calculated to check whether the requirements of Regulation (EU) No 1152/2011 had been fulfilled. Finland, Ireland and the UK carried out a representative sampling of wildlife definitive hosts present in their territory, while Malta, in the absence of wildlife definitive hosts species in its territory, did a risk based sampling of dogs. None of the four Member States who are operating an E. multilocularis-specific surveillance programme aimed at detecting the parasite, if present in any part of those Member States, has detected E. multilocularis through the surveillance activities reported in 2013. Under the assumption of unbiased representative sampling (in the case of Finland, Ireland and the UK) and unbiased risk based sampling (in the case of Malta) and considering the sensitivity of the tests applied, all four MS have fulfilled the requirement of Regulation (EU) No 1152/2011 to the effect that the surveillance activities should detect a prevalence of E. multilocularis of 1 % or less at a confidence level of at least 0.95. EFSA Journal 2013;11(11):3465 2

TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background as provided by the European Commission... 4 Terms of reference as provided by the European Commission... 4 Context of the scientific output... 4 Assessment... 5 1. Introduction... 5 2. Approach... 6 3. Assessment... 7 3.1. Finland... 7 3.2. Ireland... 11 3.3. Malta... 14 3.4. United Kingdom... 15 Conclusions and recommendations... 20 References... 20 Appendices... 22 Appendix A. Assessment tables for the surveillance report of Finland... 22 Appendix B. Assessment tables for the surveillance report of Ireland... 26 Appendix C. Assessment tables for the surveillance report of Malta... 29 Appendix D. Assessment tables for the surveillance report of the United Kingdom... 33 Appendix E. Wildlife surveillance with special emphasis on E. multilocularis... 37 Appendix F. Impact of the sampling distribution over time on the interpretation of the outcome. 38 Glossary... 40 EFSA Journal 2013;11(11):3465 3

BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION The Commission adopted Commission Regulation (EU) No 1152/2011 of 14 July 2011, as regards preventive health measures for the control of Echinococcus multilocularis infection in dogs. This was in order to ensure continuous protection of Finland, Ireland, Malta and the United Kingdom that claim to have remained free of the parasite Echinococcus multilocularis as a result of applying national rules until 31 December 2011. This Regulation includes certain obligations for these Member States to implement a pathogenspecific surveillance programme aimed at detecting the parasite, if present in any part of those Member States, in accordance with certain requirements regarding the sampling, the detection techniques and the reporting. It also provides that the Commission is to review this Regulation no later than five years following the data of its entry into force, i.e. by December 2016, in the light of scientific developments regarding EM infection in animals and submit the results of the review to the European Parliament and to the Council. Before a formal request for a scientific opinion on the infection with Echinococcus multilocularis infection in animals is addressed to EFSA to take account of the aforementioned deadline, EFSA is asked, in the context of Article 31 of Regulation (EC) No 178/2002, to provide the following scientific and technical assistance to the Commission: TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION 1. Regular follow-up of the literature regarding Echinococcus multilocularis infection in animals in the European Union and adjacent countries, including its geographical distribution and prevalence; 2. Analysis and critical assessment, in the context of Regulation (EU) No 1152/2011, of (i) the sampling strategy considered for the programmes of the Member States concerned; (ii) the data collected in the framework of these programmes; (iii) the detection methods used. EFSA is asked to produce a report regarding point 2 each year in October after reception of the Member States reports by 31 May. CONTEXT OF THE SCIENTIFIC OUTPUT This report addresses ToR 2 of the mandate M-2012-0200 submitted to EFSA by the Commission and applies the principles and procedures established in the EFSA reports Scientific and technical assistance on Echinococcus multilocularis infection in animals 4 and A framework to substantiate absence of disease: the risk based estimate of system sensitivity tool (RiBESS) using data collated according to the EFSA Standard Sample Description - An example on Echinococcus multilocularis 5. ToR 1 is being addressed by an Article 36 cooperation project of EFSA (in preparation). 4 EFSA Journal 2012;10(11):2973, 22 pp. Available online: http://www.efsa.europa.eu/en/efsajournal/pub/2973.htm 5 EFSA-Q-2012-00746. Available online: http://www.efsa.europa.eu/en/supporting/pub/366e.htm EFSA Journal 2013;11(11):3465 4

ASSESSMENT 1. Introduction Echinococcus multilocularis, a tapeworm occurring throughout the Northern Hemisphere, is transmitted primarily between wild definitive carnivorous hosts, in Europe mainly red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides), and wild intermediate hosts (small mammals, mainly arvicolid rodents, e.g. lemmings, muskrats, meadow mice and different vole species). Dogs (Canis lupus familiaris) and cats (Felis catus) can also be infected by ingestion of an intermediate host harbouring the larval form of the parasite, and may then act as definitive hosts, contributing to the persistence of the parasite in urban and periurban areas. Humans are not part of the lifecycle, but can become accidentally infected (dead-end host) by ingesting tapeworm eggs excreted by the definitive host. The resulting infection in humans, alveolar echinococcosis (AE), typically presents as an infiltrative tumour-like growth in the liver, which at later stages may invade neighbouring organs. AE is considered one of the most severe human parasitoses in non-tropical regions. In Europe, E. multilocularis is found in foxes mainly in central Europe, from the north in Denmark, the Netherlands and Belgium, in the east to the Baltic States and Slovakia, in the south to north eastern Italy and Hungary, and in the west to central France (EFSA, 2007). Recently, E. multilocularis has been identified in the red fox in Sweden (Osterman Lind, 2011). It is also present in Belarus, Ukraine and Russia, but has never been found in Finland, Ireland, Malta and the United Kingdom. EFSA was asked to analyse and critically assess the sampling strategy considered, the data collected, and the detection methods used in Member States E. multilocularis surveillance programmes in the context of Regulation (EU) No 1152/2011 regarding preventive health measures for the control of E. multilocularis infection in dogs. Regulation (EU) No 1152/2011 provides that Member States listed in Annex I thereof, i.e. Finland, Ireland, Malta and the United Kingdom, shall have had in place for the last 10 years prior to applying (a) rules for E. multilocularis infection in host animals to be compulsorily notifiable under national law; and (b) an early detection system for E. multilocularis infection in host animals. These Member States shall also implement a pathogen-specific surveillance programme which is to be drawn up and carried out in accordance with Annex II and shall report to the Commission the results of the pathogen-specific surveillance programme referred to in Annex II by 31 May following the end of each 12-month surveillance period. The objective of the pathogen-specific surveillance programme is to provide evidence for absence of E. multilocularis infection in the Member States listed in Annex I. The following requirements for the pathogen-specific surveillance programme are laid down in Annex II to Regulation (EU) No 1152/2011: 1. The pathogen-specific surveillance programme shall be designed to detect per epidemiologically relevant geographical unit in the Member State or part thereof a prevalence of not more than 1 % at confidence level of at least 95 %. 2. The pathogen-specific surveillance programme shall use appropriate sampling, either risk-based or representative, that ensures detection of the E. multilocularis parasite if present in any part of the Member State at the design prevalence specified at point 1. 3. The pathogen-specific surveillance programme shall consist in the ongoing collection, during the 12-month surveillance period, of samples from wild definitive hosts or, in the case where there is evidence of the absence of wild definitive hosts in the Member State or part thereof, from domestic definitive hosts, to be analysed by examination of: (a) intestinal contents for the detection of the E. multilocularis parasite by the sedimentation and counting technique (SCT), or a technique of equivalent sensitivity and specificity; or EFSA Journal 2013;11(11):3465 5

(b) faeces for the detection of species-specific deoxyribonucleic acid (DNA) from tissue or eggs of the E. multilocularis parasite by polymerase chain reaction (PCR), or a technique of equivalent sensitivity and specificity. Regulation (EU) No 1152/2011 applies since 1 January 2012. The first surveillance reports of the Member States listed in Annex I were therefore due by 31 May 2013. 2. Approach To address ToR 2 of the mandate submitted to EFSA by the European Commission, EFSA has developed a scientific report and a technical report in 2012 (EFSA, 2012a, b). The principles and procedures established therein have been applied in the assessment of each of the four national surveillance reports submitted to the Commission. In a first step, the description of the surveillance system was checked for completeness against the relevant elements that need to be addressed in assessing the quality of E. multilocularis surveillance reports in the context of Regulation (EU) No 1152/2011 (EFSA, 2012a). For each relevant element, (i.e. susceptible host population, timeframe of the surveillance data, relevant epidemiological unit of the surveillance system, geographical clustering of infection, case definition, sensitivity and specificity of tests used, type of survey, survey design, sampling methods, and sample size), the data provided in the surveillance report were collated (see Annex A-D), and comments were given, if needed. In a second step, the data reported on individual samples were assessed using the raw data submitted by the Member States via the EFSA Data Collection Framework (DCF). For the purpose, the software R 6 was used for descriptive statistics. Table 1 lists and describes all the parameters that were extracted from the data submitted. 6 R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.r-project.org/. EFSA Journal 2013;11(11):3465 6

Table 1: DCF List of the parameters extracted from the raw data submitted by the Member States via the Parameter Description 1 Theoretical Sampling period The twelve-months reporting period. It may go from January to December, but this is not a restriction: the reporting period can also include twelve months over 2 years. 2 Actual Sampling Period Number of days from the first sampling collection date to the last sample date within the theoretical sampling period 3 Sampling activity overtime Number of samples collected ach month within the theoretical sampling period 4 Number of samples Total number of samples collected during the theoretical sampling period 5 Number of test results Total number of test results. If the number of test results is equal to the number of samples, none of the latter required further investigations (i.e. were negative at the first test). 6 Laboratory test completion Comparison between the year at which the samples are collected and the year at which the test was completed 7 Host Target population size (N); additional information on the host species 8 Sampling Strategy and Design As reported (e.g. Objective sample Simple Random Sample) 9 Sampling point Activity adopted for the sample collection (e.g. hunting, veterinary activity, ) 10 Sampling Area Number of NUTS 3 regions covered by the sampling, number of samples per NUTS 3 region, number of samples per 1000 Km 2 11 ASe Area Sensitivity: level of confidence when stating that the actual prevalence is below the threshold foreseen in the relevant legislation (0.01 for E. multilocularis). The area sensitivity was calculated using the RiBESS tool (EFSA, 2012b) 3. Assessment 3.1. Finland In the Finnish E. multilocularis surveillance system, the coproantigen ELISA was used to screen rectal content samples collected post-mortem. The sensitivity (Se) and specificity (Sp) values reported for this test were extracted from the scientific publication of Deplazes et al. (1999) and reported as 0.836 (Se) and 0.995 (Sp). Confirmation of ELISA-positive samples was carried out by Sedimentation and Counting Technique (SCT). The overall test sensitivity of this diagnostic approach is reported as 0.836 x 0.99 = 0.828. However, if expressed relative to the gold standard diagnostic technique, the SCT, which has 99 % Se and 100 % Sp, the Se of the coproantigen ELISA is 80 %, while the Sp is 95-99 % (EFSA, 2012a). Using this conservative estimate of the ELISA Se, results in an overall test sensitivity of 0.792 of this diagnostic approach. The susceptible wild definitive host population targeted by the system was defined as raccoon dogs and red foxes. The justifications reported for choosing these target species were the facts that the red fox is the primary host of E. multilocularis in Europe, that raccoon dogs have been shown to be good definitive hosts for E. multilocularis and that raccoon dogs are more numerous in Finland than red foxes. The epidemiological unit was defined as the individual animal. The size of the raccoon dog population is reported to be 230,000 animals, the red fox population reportedly encompassed 150,000 animals. These figures are based on a Finnish study from 2007, which estimated the average maximum size of the red fox and raccoon dog populations in autumn, combining information from radio-telemetry studies, annual snow track counts (foxes), hunting bag records and breeding capacity (Kauhala, 2007). The distribution of the red fox and raccoon dog populations is reported to vary across EFSA Journal 2013;11(11):3465 7

the country with raccoons being most numerous in the Southeast and South Finland and fox density being highest in the South and Southwest Finland, according to the same study (Kauhala, 2007). The sampling strategy is reported as providing a representative sample. The animal samples were collected by hunters. Information on zoonoses surveillance and sampling was disseminated to local hunting associations in order to activate hunters of small game to submit foxes and raccoon dogs to the authorities. The sample size has been calculated using an overall sensitivity of the diagnostic approach of 0.828 and the design prevalence of 1 % prescribed in Regulation (EU) No 1152/2011 using the RiBESS tool. 234 and 259 samples were collected from foxes and raccoon dogs respectively. 480 samples were negative in the coproantigen ELISA test, 13 samples which tested positive in the ELISA were found to be negative in the SCT. Thus, no sample tested was found positive for Echinococcus multilocularis. The sampling covered 0.16 % of the reported red fox population and 0.11 % of the reported raccoon dog population (0.13 % of the targeted population of susceptible wildlife hosts reported). Samples were collected throughout the 12 months surveillance period of 2012 (Figure 1) from 17 of the 20 Finnish NUTS 3 regions (Figure 2). The highest number of samples was collected in January (n=133), the lowest number in June (n=4). The reason for the temporal distribution of the sample collection is reported as the restrictions to hunt females with pups during the months of May, June and July. The spatial distribution of the samples collected indicates that not all NUTS 3 regions have been sampled with the same intensity (Figure 2). It is reported that 2 NUTS 3 regions were not sampled in 2012 due to a lack of voluntary hunters. It is reported that the main emphasis of sample collection was placed on the Southeast region bordering Russia and Estonia, for which a sampling goal had been set by the veterinary authorities, because the risk of introduction by natural means was considered to be highest from those countries. This region is also reported to have the highest density of raccoon dogs. Approximately 50% of all samples reported were from the 5 NUTS regions bordering these countries (79 % of raccoon dog samples, 18% of red fox samples) (Figure 2). The highest density of the fox population is reported to be in the Southwest. It is stated that in future years, more effort will be placed on achieving a more representative sample of the red fox population by increasing the sample collection in the South and Southwest of Finland. Using the RiBESS tool under the assumption of a representative sample and considering the more conservative test sensitivity of 0.792, the value for the area sensitivity was calculated to be 0.98. This means that the requirement of Regulation (EU) No 1152/2011 regarding a confidence level of at least 0.95 has been achieved. For future reports, considerations regarding potential biases inherent in the survey design and assumptions and uncertainties related to the sampling methods should be provided. In summary, the set of data provided for the year 2012 gives an adequate area sensitivity in line with the requirements of Regulation (EU) No 1152/2011. Considering the sample size and the target population size, the minimum test sensitivity needed to obtain an adequate area sensitivity is 0.6 (60%). EFSA Journal 2013;11(11):3465 8

140 130 120 110 100 90 p 80 70 60 50 40 30 20 10 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2012 Figure 1: Finland - Sampling activity by month of sampling EFSA Journal 2013;11(11):3465 9

Number of samples per NUTS3 region!!! 1-10 11-25 26-50 51-100 101-293 No samples!!!!!!!!!!!!!!!!!!! Sampling Intensity (Samples per 1000 km2) 0.04-0.57 0.58-1.66 1.67-3.9 4-9.5 9.6-2045 No samples!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Figure 2: Finland - Sampling activity and intensity by NUTS 3 region EFSA Journal 2013;11(11):3465 10

3.2. Ireland The modified SCT (Hofer et al., 2000) was used in the Irish E. multilocularis surveillance system to analyse intestinal contents of individual animals. This technique is considered to have a Se and Sp of approximately 100 %. For the calculation of the sample size, values of 99.5 % Se and Sp were used by the Irish authorities. However, it has been suggested by Eckert to consider a Se of 98 % to take account of potential individual errors (Eckert, 2003). The susceptible wild definitive host population targeted by the system was defined as red foxes, based on the red fox being a potential wildlife definite host species distributed throughout Ireland and raccoon dogs never having been reported to occur in the country. The epidemiological unit was defined as the individual animal. The size of the red fox population reportedly encompassed 150,000 animals. This figure was based on a study, which estimates the breeding population to be between 150,000 and 200,000. In this study, it is also stated that foxes are distributed throughout Ireland (Hayden and Harrington, 2000). The sampling strategy is reported as providing a representative sample. The animal samples were obtained through hunting. It is reported that each of the 16 Regional Veterinary Offices in Ireland was requested to obtain a number of wild foxes, based on the number of counties that a specific office regulated, and such that the sum of the target regional numbers of foxes would yield the required total number of samples nationally. Each Regional Office contacted hunting societies in their area and asked them to inform hunters to submit shot foxes to the nearest Regional Veterinary Laboratory for collection of fox intestines. The design prevalence of 1 % prescribed in Regulation (EU) No 1152/2011 and a test Se of 0.995 were used in the sample size calculation. The collection of 324 samples is reported by Ireland. All 324 samples are reported as having tested negative for E. multilocularis using the modified SCT. The sampling covered 0.22 % of the reported red fox population. Samples were collected mainly during the months October, November and December 2012, with an additional sample collected in January 2013 (Figure 3). The reason that is reported for this temporal distribution of sampling was the aim to avoid culling adult female foxes with fox cubs dependant on their dam fox for food. It is reported that collection of samples during the winter months only would not adversely affect the sensitivity of the survey, based on a study from an endemic urban area in Switzerland, which found a greater prevalence of E. multilocularis in foxes in winter months (Hofer et al., 2000). Samples were collected in all 8 Irish NUTS 3 regions (Figure 4). The Border region accounts for 29 % of the samples, the South-East region for 19%, while approximately 10 % of samples come from the other regions (exception: Dublin with only 1 sample). Using the RiBESS tool under the assumption of a representative sample and considering the more conservative test sensitivity of 0.98, the area sensitivity has been calculated as 0.96 (96%). This means that the requirement of Regulation (EU) No 1152/2011 regarding a confidence level of at least 0.95 has been achieved. For future reports, considerations regarding potential biases inherent in the survey design and assumptions and uncertainties related to the sampling methods should be provided. In summary, the set of data provided for the year 2012 gives an adequate area sensitivity in line with the requirements of Regulation (EU) No 1152/2011. Considering the sample size and the target population size, the minimum test sensitivity value needed to obtain an adequate area sensitivity is 0.925 (92.5%). EFSA Journal 2013;11(11):3465 11

150 140 130 120 110 Number of Samples 100 90 80 70 60 50 40 30 20 10 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2012 Figure 3: Ireland - Sampling activity by month of sampling EFSA Journal 2013;11(11):3465 12

Number of samples per NUTS3 region 1-10 11-25!!! 26-50 51-100 101-293 No samples!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Sampling Intensity (Samples per 1000 km2) 0.04-0.57 0.58-1.66 1.67-3.9 4-9.5 9.6-2045 No samples!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Figure 4: Ireland - Sampling activity and intensity by NUTS 3 region EFSA Journal 2013;11(11):3465 13

3.3. Malta In the Maltese E. multilocularis surveillance system, the Copro-DNA detection by PCR was used to analyse faecal samples of live animals (Mathis et al., 1996). First, faecal samples were tested for the presence of taeniid eggs using the flotation method. Where eggs were detected, these samples were submitted to the EU Reference Laboratory for Parasites for species identification by PCR. The combined use of flotation and PCR as described by Mathis et al. (1996) was considered to have a specificity of 100 % and a sensitivity of 94 % compared to the SCT (Eckert et al., 2003). The susceptible definitive host population targeted by the system was defined as dogs. This was justified by the reported absence of the known wild definitive host species from Malta. Information available from the International Union for Conservation of Nature and Natural Resources has been cited as evidence for this. The epidemiological unit was defined as the individual live animal. The size of the dog population was estimated to be 60,000 animals, with 2,000 dogs being reported as non-pet dogs. Subpopulations included in the surveillance system are hunting dogs, dogs in sanctuaries and rural dogs on farms, chosen due to their putative exposure to infective intermediate host animals. The size of the rural dog population was estimated to be 4,500 animals by assuming 2 dogs per farm in Malta. The estimate is reported to be supported by information available from NGOs offering neutering and microchiping services to farmers. The reported size of the stray dog population of 2,000 animals was based on records available at the six animal sanctuaries of Malta, while the estimate of the hunting dog population size of 10,000 animals is reported to be based on a neutering campaign having been carried out in 2009. Due to the small geographic area of Malta, the distribution of the susceptible population is reported as not being relevant. It is stated that 320 samples were planned to be collected, encompassing 100 samples each from hunting and rural dogs and 120 samples from sanctuary dogs. It is not explained how the sample size has been calculated and no scientific reasoning for the distribution of the sample across the subpopulations is provided. The sampling strategy is reported as being a risk based surveillance. The susceptible host population has been stratified into 3 categories (stray, hunting and rural (farm) dogs) considered to be at risk for E. multilocularis infection due to their potential exposure to infected intermediate hosts. Two risk indicators have been identified. The indicator exposition to intermediate hosts was divided into 2 ranges, less exposed and exposed. The indicator importation from non-free areas was divided into 2 ranges, unknown and known. The relative risk associated with exposure to intermediate host was estimated as 1.2, the relative risk associated with unknown importation from non-free areas was estimated as 1.2. No evidence or reasoning for the RR of 1.2 for unknown importation from non-free areas has been provided. The references cited as the basis for the RR of 1.2 for exposure to intermediate host give an odds ratio (OR) of 4.28 for use as hunting dog and 0.39 for being tied up at all times (Ziadinov et al., 2008) and an OR of 6.36 for use as shepherd dog, an OR of 7.05 for being fed raw viscera and an OR of 6.09 for catching rodents (Antolova et al., 2009). Estimates of the proportion of the population allocated to each risk indicator category are provided. They are based on the estimates of the stray, hunting and farm dog populations. It is reported that all risk areas have been considered. A total of 293 samples were obtained from hunting dogs (n=42), dogs in sanctuaries (n=133) and from rural dogs (n=118). All samples are reported to have been tested by the flotation technique. The flotation technique identified 24 of the 293 samples as positive for taeniid eggs. These 24 samples were further analysed by PCR and found to be negative for E. multilocularis. The sampling covered 0.42 % of the reported hunting dog population, 6.65% of the reported stray dog population and 2.62% of the reported rural dog population. Samples were collected from March to December 2012, with the majority of samples having been collected from September to December (Figure 5), which coincides with the Maltese hunting season (Sep-Jan). An ascertainment of the recent deworming history has been carried out only for stray dogs sampled at sanctuaries, not for the sampled rural and hunting dogs. It is not reported whether the time since EFSA Journal 2013;11(11):3465 14

arrival at the sanctuary has been established for sampled stray dogs. No discussion of how this could bias the results is provided. Using the RiBESS tool under the assumption of a risk based sample and considering the test sensitivity of 0.94, the area sensitivity has been calculated as 0.988 (98.8%). This means that the requirement of Regulation (EU) No 1152/2011 regarding a confidence level of at least 0.95 has been achieved. For future reports, considerations regarding potential biases inherent in the survey design and assumptions and uncertainties related to the sampling methods should be provided. In summary, the set of data provided for the year 2012 gives an adequate area sensitivity in line with the requirements of Regulation (EU) No 1152/2011. Considering the sample size and the target population size, the minimum test sensitivity value needed to obtain an adequate area sensitivity is 0.636 (63.6%). 60 50 Number of Samples 40 30 20 10 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2012 Figure 5: Malta - Sampling activity by month of sampling 3.4. United Kingdom In Northern Ireland, the SCT was used to analyse intestinal contents of individual animals collected post-mortem. The method was considered to have a specificity of 100 % and a sensitivity of 99 % (Eckert et al., 2001). In Great Britain (England, Scotland and Wales) (GB), a PCR test was used to detect E. multilocularis DNA in faeces by rectal sampling (Mathis et al., 1996; Dinkel et al., 1998). The test was considered to have 0.94 Se and 1.0 Sp compared to the gold standard SCT (Eckert, 2003). The susceptible wild definitive host population targeted by the system was defined as red foxes. It is reported that no other wildlife definitive host species exists in the UK, however, no reference is provided for the reported absence of other wildlife definitive host species. The epidemiological unit was defined as the individual animal for the sampling points for road kills and hunting, and faecal contents from rectal samples from hunted animals for the sampling point wildlife research stations. The size of the red fox population reportedly encompassed 250,000 animals in GB and 14,000 in Northern Ireland. The population size estimate for GB was based on a survey carried between 02/1999 and 03/2000, which estimated the breeding population of adult rural foxes and urban foxes as 258,000 EFSA Journal 2013;11(11):3465 15

(Confidence Interval 212,000-304,000) (Webbon et al., 2004). More recent, unpublished data is reported to indicate that these figures have not changed substantially. No evidence is provided for the population figures from Northern Ireland. The distribution of the red fox population in GB is reported using a figure from the 1999/2000 survey (Figure 6). Figure 6: Estimated rural fox density per square kilometre in Great Britain (excluding revised estimates based on urban areas). Source: Webbon et al., 2004. The sampling strategy is reported as providing a representative sample. Due to the geographical separation of Northern Ireland from GB, two distinct host populations have been used in the survey design. The samples were obtained from wildlife research stations, through hunting and from road kills in GB, while in Northern Ireland, samples were obtained through hunting and road kills only. It is reported that the number of foxes to be tested per area was calculated based on the estimated population density. Collected samples were mapped and home based staff was contacted towards the end of the sampling period to collect any missing samples. The sample size has been calculated using values of 0.85 Se and 1.0 Sp (PCR) for GB and using 0.99 Se and 1.0 Sp (SCT) for Northern Ireland and the design prevalence of 1 % prescribed in Regulation (EU) No 1152/2011. A total of 701 samples were obtained from wildlife research stations (57 %), through hunting (39 %) and from road kills (4 %). All 398 samples from GB analysed by PCR and all 303 samples from Northern Ireland analysed by SCT tested negative for E. multilocularis. The sampling covered 0.2 % of the reported red fox population of GB (n=398) and 2.2 % of the reported red fox population of Northern Ireland (n=303). Samples were collected from October 2012 to February 2013 in GB and from September 2012 to March 2013 in Northern Ireland (Figure 7 and 8). This temporal distribution of the sampling results from the intention to avoid hunting foxes with cubs depending on their dams for food during the months of March to August. Samples were collected in from 38 of the 133 UK NUTS 3 regions (Figure 9). It is reported that for some areas the number of samples collected was small due to balancing costs, practicality and robustness of data. EFSA Journal 2013;11(11):3465 16

Using the RiBESS tool under the assumption of a representative sample and considering a test sensitivity equal to 0.85, the area sensitivity of the surveillance system of GB has been calculated as 0.97 (97%). For the surveillance system of Northern Ireland, using the RiBESS tool under the assumption of a representative sample and considering a test sensitivity equal to 0.99, the area sensitivity has been calculated as 0.95 (95%). This means that the requirement of Regulation (EU) No 1152/2011 regarding a confidence level of at least 0.95 has been achieved for both systems operated in the UK. For future reports, considerations regarding potential biases inherent in the survey design and assumptions and uncertainties related to the sampling methods should be provided. In summary, it must be specified that the submitted data from the United Kingdom (England, Scotland and Wales and Northern Ireland) covers more than a 1 year period. As the relevant legislation foresees a 12 months reporting period, two analyses were carried out: i) a first one considering only the samples collected in 2012 and ii) a second one considering a period going back up to 12 months starting from the date at which the last sample was collected. The first set of data (i) did not allow reaching an area sensitivity in line with the relevant legislation (95%). For this reason, this assessment was performed based on the second set of data, calculated as follows: last sample collection date: 19th February 2013, last sample collection month: February 2013; beginning of the sampling period: March 2012 (i.e. 12 months before). Calculated as specified above, the sampling provides an adequate area sensitivity in line with the requirements of Regulation (EU) No 1152/2011. Thus, the next reporting period should be from the 1st March 2013 to February 2014. Considering the sample size and the target population size for GB, the minimum test sensitivity value needed to obtain an adequate area sensitivity would be 0.745 (74.5%). Considering the sample size and the target population size for NI, the minimum test sensitivity value needed to obtain an adequate area sensitivity would be 0.968 (96.8%). 140 130 120 110 100 90 80 p 70 60 50 40 30 20 10 0 2012 2013 Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 2012 Figure 7: Great Britain - Sampling activity by month of sampling EFSA Journal 2013;11(11):3465 17

60 55 50 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Number of Samples 45 40 35 30 25 20 15 10 5 0 2012 2013 Figure 8: Northern Ireland - Sampling activity by month of sampling EFSA Journal 2013;11(11):3465 18

Number of samples per NUTS3 region!!! 1-10 11-25 26-50 51-100 101-293 No samples!!!!!!!!!! Sampling Intensity (Samples per 1000 km2) 0.04-0.57 0.58-1.66 1.67-3.9 4-9.5 9.6-2045 No samples!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Figure 9: United Kingdom - Sampling activity and intensity by NUTS 3 region EFSA Journal 2013;11(11):3465 19

CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS None of the four Member States who are operating an E. multilocularis-specific surveillance programme aimed at detecting the parasite, if present in any part of those Member States, has detected E. multilocularis through the surveillance activities reported in 2013. Under the assumption of an unbiased representative sample (in the case of Finland, Ireland and the UK) and an unbiased risk based sample (in the case of Malta) and considering the sensitivity of the tests applied, all four MS have fulfilled the requirement of Regulation (EU) No 1152/2011 regarding detecting a prevalence of not more than 1 % with a confidence level of at least 0.95. Surveillance of wildlife populations for the presence of E. multilocularis presents many challenges (see Appendix E). Establishing the size of the target wildlife population is resource-consuming; identifying the location of the animals to be sampled is considerably more difficult than in the case of domestic animals. The MS have used a robust approach to these challenges by basing their sample size calculations on available population size estimates and by distributing the sampling over their entire territory. Appendix E also contains information about the different potential biases that have been reported to occur with wildlife samples collected through hunting, road-kill and other convenient and frequently applied methods. Such considerations might be important for the four countries to include when designing their sampling strategies to minimise the potential negative influences of such biases. A skewed distribution of the sampling activity over time, as reported by several MS, may not be an issue of concern in this specific framework, as has been illustrated in Appendix F. In fact, one could argue that if E. multilocularis was introduced towards the end of the surveillance period of 12 months and assuming an even time distribution of sampling, then the confidence in detecting an infection slightly above 1% is likely to be lower than 0.95, due to fewer samples collected during the actual risk period. But this may be considered more as a question of missed early detection, since the infection should be picked up by the surveillance with the required efficacy during the following year. RECOMMENDATIONS For future reports, some considerations regarding potential biases inherent in the survey design and the assumptions and uncertainties related to the sampling methods should be provided in the surveillance reports with the aim to identify how the surveillance can be further developed or adapted. Special emphasis should be placed on how to minimise potential biases from using convenient sources of wildlife samples, as well as to reflect the potential clustering of infection across time, space and host age groups (see Appendices E and F). REFERENCES Antolova D, Reiterova K, Miterpakova M, Dinkel A and Dubinsky P, 2009. The First Finding of Echinococcus multilocularis in Dogs in Slovakia: An Emerging Risk for Spreading of Infection. Zoonoses and Public Health, 56, 53 58. Conraths FJ, Staubach C and Tackmann K, 2003. Statistics and sample design in epidemiological studies of Echinococcus multilocularis in fox populations. Acta Tropica, 85, 183-189. Deplazes P, Alther P, Tanner I, Thompson RCA and Eckert J, 1999. Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations. Journal of Parasitology, 85, 115-121. Dinkel A, Nickisch-Rosenegk M, Bilger B, Merli M, Lucius R and Romig T, 1998. Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy. Journal of Clinical Microbiology, 36, 1871 1876. EFSA Journal 2013;11(11):3465 20

Eckert J, Gemmell MA, Meslin FX and Pawlowski ZS, 2001. WHO/OIE Manual on Echinococcosis in humans and animals: a public health burden of global concern. World Organisation for Animal Health, Paris. Eckert J, 2003. Predictive values and quality control of techniques for the diagnosis of Echinococcus multilocularis in definitive hosts. Acta Tropica, 85, 157-163. EFSA (European Food Safety Authority), 2007. Scientific opinion of the Panel on Animal Health and Welfare (AHAW Panel) regarding the assessment of the risk of Echinococcosis introduction into the UK, Ireland, Sweden, Malta and Finland as a consequence of abandoning national rules. The EFSA Journal 2007, 441, 1-59. doi:10.2903/j.efsa.2007.441 EFSA (European Food Safety Authority), 2012a. Scientific and technical assistance on Echinococcus multilocularis infection in animals. EFSA Journal 2012;10(11):2973, 22 pp. doi:10.2903/j.efsa.2012.2973 EFSA (European Food Safety Authority), 2012b. A framework to substantiate absence of disease: the risk based estimate of system sensitivity tool (RiBESS) using data collated according to the EFSA Standard Sample Description An example on Echinococcus multilocularis. Supporting Publications 2012:EN-366, 44 pp. Hayden T and Harrington R, 2000. Exploring Irish mammals. Town House & Country House Ltd., Dublin, Ireland. Hofer S, Gloor S, Müller U, Mathis A, Hegglin D and Deplazes P, 2000. High prevalence of Echinococcus multilocularis in urban red foxes (Vulpes vulpes) and voles (Arvicola terrestris) in the city of Zürich, Switzerland. Parasitology, 120, 135-142. Johnson DH, 2002. The Importance of Replication in Wildlife Research. Journal of Wildlife Management 66, 919-932. Kauhala K, 2007. Paljonko Suomessa on pienpetoja? Riista-ja kalatalous Selvityksiä 1, Riista-ja kalatalouden tutkimuslaitos, Helsinki 2007. Mathis A, Deplazes P and Eckert J, 1996. An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs. Journal of Helminthology, 70, 219-222. Nusser SM, Clark WR, Otis DL and Huang D, 2007. Sampling considerations for disease surveillance in wildlife populations. Journal of Wildlife Management, 72, 52-60. Osterman Lind E, Juremalm M, Christensson D, Widgren S, Hallgren G, Ǻgren EO, Uhlhorn H, Lindberg A, Cedersmyg M and Wahlström H, 2011. First detection of Echinococcus multilocularis in Sweden, February to March 2011. Euro Surveillance, 16, pii=19836. Staubach C, Thulke HH, Tackmann K, Hugh-Jones M and Conraths FJ, 2001. Geographic Information System-aided analysis of factors associated with the spatial distribution of Echinococcus multilocularis infections of foxes. American Journal of Tropical Medicine and Hygiene, 65, 943-948. Tackmann K, Löschner U, Mix H, Staubach C, Thulke HH and Conraths FJ, 1998. Spatial distribution patterns of Echinococcus multilocularis (Leuckart 1863) (Cestoda: Cyclophyllidea: Taeniidae) among red foxes in an endemic focus in Brandenburg, Germany. Epidemiology and Infection, 120, 101-109. Webbon CC, Baker PJ and Harris S (2004). Faecal density counts for monitoring red fox numbers in rural Britain. Journal of Applied Ecology, 41, 768-779. Ziadinov I, Mathis, A, Trachsel D, Rysmukhambetova A, Abdyjaparov TA, Kuttubaev OT, Deplazes P and Torgerson PR, 2008. Canine echinococcosis in Kyrgyzstan: Using prevalence data adjusted for measurement error to develop transmission dynamics models. International Journal for Parasitology, 38, 1179 1190. EFSA Journal 2013;11(11):3465 21

APPENDICES Appendix A. Assessment tables for the surveillance report of Finland Table 2: Assessment of the description of the surveillance system (Part I of surveillance report) for a representative sample survey Element Description of Element Information provided in surveillance report Comments DP is specified in Annex II to Regulation The design prevalence of 1 % prescribed in the Regulation (EU) No 1152/2011and must be 1 % or None was used in the sample size calculation. lower. Design Prevalence (DP) Test sensitivity Definition of susceptible host population targeted by the system Epidemiological unit The sensitivity of the test used in the surveillance system must be reported. This would ideally be estimates from each participating laboratory reported as a point estimate (average) of the values across the country with minimum and maximum values or a probability distribution. Alternatively, reference to estimates published in peerreviewed scientific literature can be made. The susceptible wild definitive host population(s) (red foxes, raccoon dogs) targeted by the surveillance system should be described and the choice justified. If domestic host species (dogs or cats) are sampled, evidence for the absence of wild definitive hosts and for these domestic animals having had access to outdoors should be provided. It should be clearly defined if individual animals or individual faeces samples collected from the environment constitute the epidemiological unit. If individual faeces samples are collected from the environment, the method applied to establish the species from which the faeces originated has to be reported. The coproantigen ELISA was used to screen rectal content collected post-mortem. The sensitivity (Se) and specificity (Sp) values reported for this test were extracted from the scientific publication of Deplazes et al. (1999) and reported as 0.836 (Se) and 0.995 (Sp). Confirmation of ELISApositive samples was carried out by Sedimentation and Counting Technique (SCT). The overall test sensitivity is reported as 0.836 x 0.99 = 0.828. The susceptible wild definitive host population targeted by the system was defined as raccoon dogs (Nyctereutes procyonoides ) and red foxes (Vulpes vulpes). The justifications for choosing these target species were the fact that the red fox is the primary host of E. multilocularis in Europe, that raccoon dogs have been shown to be good definitive hosts for E. multilocularis and that raccoon dogs are more numerous in Finland than red foxes. The epidemiological unit was defined as the individual animal. The animals were provided to the authorities by hunters. If expressed relative to the gold standard diagnostic technique, the SCT, which has 99 % Se and 100 % Sp, the Se of the coproantigen ELISA is 80 %, while the Sp is 95-99 % (EFSA, 2012a). If using this more conservative Se estimate, the overall sensitivity of the diagnostic approach was 0.800 x 0.99 = 0.792. None None EFSA Journal 2013;11(11):3465 22