Technical specifications for monitoring Community trends in zoonotic agents in foodstuffs and animal populations 1

SCIENTIFIC REPORT OF EFSA Technical specifications for monitoring Community trends in zoonotic agents in foodstuffs and animal populations 1 European Food Safety Authority 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT Technical specifications are proposed for the monitoring of temporal trends in zoonotic agents in animal and food populations at Community or Member State group level in the framework of Directive 2003/99/EC. Two types of trend monitoring are identified: trend watching, which covers general observations of harmonised or non-harmonised data for possible trends, and trend analyses which means statistical analyses of harmonised data for the detection of trends over time. Trend watching can be regarded as the first and preliminary step in trend monitoring. The specifications identify a set of criteria for the selection of the zoonotic agent/animal or food category combinations where trend analyses would be justified. Based on data available from 2004 to 2007, the following combinations are suggested for trend analyses: Salmonella in fresh broiler and pig meat, flocks of laying hens and broilers, slaughter and breeding pigs as well as fattening turkeys; Campylobacter in fresh broiler meat; Listeria monocytogenes in smoked fish; Mycobacterium bovis in bovine herds; Brucella in bovine and caprine/ovine herds in co-financed non-officially free Member States; verotoxigenic Escherichia coli O157 in cattle; and Echinococcus multilocularis in red foxes. This list is proposed to be revised on a regular basis taking into account most recent knowledge. Suggestions for minimum sample sizes and number of time points needed for identifying trends are provided. Weighting of the national results should be applied at Community level in order to account for the different sizes of national populations. Biological relevance of trends depends on several factors such as the prevalence of the agent in the population, and the severity of the disease in question, as well as on the impact of the trend on the exposure to humans. Statistical significance of a trend observed in data is therefore only one of the factors impacting on the biological relevance of an observed trend. KEY WORDS Animal population, foodstuff, trend analysis, sample size, zoonotic agent. 1 On request of EFSA, Question No EFSA-Q-2008-263, issued on 28 February 2010. 2 Correspondence: zoonoses@efsa.europa.eu. 3 Acknowledgement: EFSA wishes to thank the members of the Task Force on Zoonoses Data Collection that endorsed this report: Andrea Ammon, Lisa Barco, Marta Bedriova, Susan Chircop, Georgi Chobanov, Adriana Costache, Jürg Danuser, Noel Demicoli, Kris De Smet, Sylvie Francart, Matthias Hartung, Birgitte Helwigh, Merete Hofshagen, Simona Iannetti, Sarolta Idei, Patrícia Inácio, Helle Korsgaard, Elina Lahti, Lesley Larkin, Peter Much, Edith Nagy, Lisa O Connor, Rob Van Oosterom, Jacek Osek, Manca Pavšič, Melanie Picherot, Christodoulos Pipis, Saara Raulo, Tatiana Ribakova, Jose Luis Saez Llorente, Petr Šatrán, Snieguole Sceponaviciene, Joseph Schon, Jelena Sõgel, Ana María Troncoso González, Kilian Unger, Luc Vanholme, Dimitris Vourvidis. Also the contribution of the members of the Working Group on Trends in Zoonotic Agents in Animals and Food that prepared this scientific report is gratefully acknowledged: Apostolos Angelidis, Birgitte Borck, Mariann Chriél, Matthias Hartung, Ari Horman, Sarolta Idei, Helle Korsgaard and Maria Luisa Pulido Puente and EFSA s staff members Pia Makela, Valentina Rizzi, Billy Amzal and Frank Boelaert for the support provided to this EFSA scientific report. For citation purposes: European Food Safety Authority; Technical specifications for monitoring Community trends in zoonotic agents in foodstuffs and animal populations on request from EFSA.. [45 pp.]. doi:10.2903/j.efsa.2010.1530. Available online: www.efsa.europa.eu. European Food Safety Authority, 2010 1

SUMMARY The Directive 2003/99/EC lays down the Community system for the monitoring and collection of information on zoonoses, which obliges Member States to collect relevant, and where applicable, comparable data on zoonoses, zoonotic agents, antimicrobial resistance and food-borne outbreaks. With the aim of developing the monitoring of trends over time in zoonotic agents, the Task Force on Zoonoses Data Collection was asked by the European Food Safety Authority to identify for which zoonotic agent/animal population or food category combinations the monitoring of trends would be useful either at Community or Member State group level. In these technical specifications two types of trend monitoring are identified: trend watching, which covers the general observation of harmonised or non-harmonised data for possible temporal trends, and trend analyses which means statistical analyses of harmonised data for the detection of trends over time. The difference between trend watching and trend analyses is in the strength of the evidence identifying the existence of trend over years. Trend watching gives indications where temporal trends may occur without making statements if the apparent trend is likely to be a real one or not. Trend watching can be regarded as the first and preliminary step in trend monitoring. A set of criteria are suggested for selecting the zoonotic agent/animal or food category combinations where trend analyses would be justified at Community or Member State group level. These criteria are based on public health importance and other needs for close monitoring of situations at Community level. The application of the criteria may result in different outcomes when applied at different time points. Based on data available from 2004 to 2007, the following fields are suggested suitable for trend analyses: Salmonella in fresh broiler and pig meat, flocks of laying hens and broilers, slaughter and breeding pigs as well as fattening turkey flocks; Campylobacter in fresh broiler meat; Listeria monocytogenes in smoked fish; Mycobacterium bovis in herds of co-financed non-officially bovine tuberculosis free Member States; Brucella in bovine and caprine/ovine herds of co-financed nonofficially brucellosis free Member States; Verotoxigenic Escherichia coli O157 in cattle; and Echinococcus multilocularis in red foxes. It is proposed that this list be revised on a regular basis taking into account new scientific knowledge regarding zoonotic agents, their prevalence in different categories of food/animal populations, their incidence in humans and also based on new Community legislation. The technical specifications also provide the rationale and suggestions for minimum sample sizes that will enable the detection of a trend after a number of time points (e.g. years). Based on a simulation carried out, it appears that when the starting (baseline) prevalence at Community or Member State group level is high (50%) and decreases by 10% per time point, the trend at European Union level or at the level of a group of Member States can be observed. A minimum Member State-specific sample size is required of 245 taken twice (at two time points) after the baseline time point or with a minimum sample size of 65 taken three times (at three time points) after the baseline time point. In the case of a moderate starting prevalence (10%) decreasing by 10% in time, higher sample sizes are required. In the scenario of a low starting prevalence of 2% that decreases by 10%, substantially more samples are needed and only after five to six time points, reasonable sample sizes, respectively 615 and 375, would enable the detection of the decreasing trend. The weighting of national results should be applied at Community level in order to account for the different sizes of national production and animal populations. The statistical significance of the trend observed is only one of the factors impacting on the biological relevance of the observed trend. Biological relevance of trends depends on several factors such as the prevalence of the agent in the population, the severity of the disease in question, as well as on the impact of the trend on the exposure of humans to the agent. 2

TABLE OF CONTENTS Abstract... 1 Summary... 2 Background as provided by EFSA... 4 Terms of reference as provided by EFSA... 4 Technical specifications... 5 1. Introduction... 5 2. Current data collection and trend analyses... 6 2.1. Current data collection system... 6 2.2. Data characteristics... 7 2.3. Current trend watching and analysis... 9 3. Rationale for monitoring the trends and criteria for selection of the fields where to monitor trends... 12 3.1. Types of trend monitoring... 12 3.2. Criteria to select fields where to analyse trends... 13 4. Fields where to analyse trends... 15 5. Statistical aspects of trend analyses minimum required sample size... 17 5.1. Periodic sample size at Member State level enabling trend detection at EU level... 17 6. Biological relevance of trends in zoonotic agents in food and animal populations... 22 7. Weighting the results for trend analyses... 22 7.1. Rationale and importance of weighting... 23 7.2. Weighting factors to be used... 23 8. Needs for the harmonisation of monitoring and reporting... 25 Conclusions... 27 References... 29 Appendices... 30 A. Statistical analysis of temporal trends of zoonotic agents in animals and food... 30 B. Application of the selection criteria of the zoonotic agent/food-animal categories where trends are to be analysed... 31 C. General aspects of sample size calculations... 35 D. Simulation-based approach to calculate the sample size required to detect significant trends... 40 E. Definitions... 42 Abbreviations... 45 3

BACKGROUND AS PROVIDED BY EFSA Directive 2003/99/EC 4 of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC, lays down the Community system for the monitoring and reporting of information on zoonoses, which obligates Member States (MSs) to collect relevant, and where applicable, comparable data on zoonoses, zoonotic agents, antimicrobial resistance and food-borne outbreaks. The European Food Safety Authority (EFSA) is assigned the tasks of examining the data collected and preparing the Community Summary Report (CSR). The Directive asks EFSA to publish a CSR on trends and sources of zoonoses, zoonotic agents, antimicrobial resistance and food-borne outbreaks. The following of trends at Community and MSspecific levels is important because it provides information on emerging or re-emerging zoonoses and it is also a tool to evaluate whether the Community or national control measures are effective in reducing the occurrence of zoonotic agents. A substantial amount of data is reported each year on zoonotic agents in a wide range of different animal species and food categories, and there is a need to specify where the following of trends would be most important. In the analyses of trends, EFSA applies epidemiological and statistical methods and the data available from MSs is not always sufficiently complete to enable a proper trend analysis. EFSA is in the process of developing statistical analyses of trends in respect of the most appropriate analyses methods to be applied. TERMS OF REFERENCE AS PROVIDED BY EFSA The Task Force on Zoonoses Data Collection is asked to: consider for which zoonotic agent/animal population or food category combinations the following of trends over time would be useful in the CSR, either at MS-specific and/or Community level; specify what information on the sample or sampling should be provided by MSs to enable an optimal analysis; evaluate the biological relevance of trends in addition to their statistical significance; issue a report on the following of trends over time in zoonotic agents in foodstuffs and animal populations; provide recommendations for the further development of reporting and monitoring in view of improved trend analyses. 4 OJ L 325, 12.12.2003 p. 31. 4

TECHNICAL SPECIFICATIONS 1. Introduction Data collected in the framework of Directive 2003/99/EC relate to the occurrence of zoonotic agents in animals, food and feed, as well as to antimicrobial resistance in these agents. The information concerning zoonoses cases in humans and related antimicrobial resistance is derived from the communicable disease networks that are currently coordinated by the European Centre for Disease Prevention and Control (ECDC). EFSA, in close collaboration with ECDC, has published five CSRs on Trends and Sources of Zoonoses, Zoonotic Agents, Antimicrobial Resistance and Food-borne Outbreaks in the European Union (EU), the latest on data for 2008. EFSA is assisted by its Zoonoses Collaboration Centre (ZCC) in the drafting of the report. In the CSR, data submitted by MSs are analysed using descriptive and analytical epidemiological methods. Also, spatial (geographical) analyses are used when appropriate. It is acknowledged that, in most cases, data received in the framework of the annual zoonoses data collection are not directly comparable between countries. This is due to differences in the sampling strategies and analytical methods applied. However, it is assumed that in most cases data are relatively comparable between reporting years within the countries, unless the monitoring systems have been substantially changed. Thus, the following of trends in the occurrence of agents is the preferred option in data analyses. The following of trends both at Community and MS group levels provides information on developments in the Community and possible re-emergences of the zoonoses monitored. It is also a tool to indirectly evaluate whether the Community or national control measures are effective in reducing the occurrence of the zoonotic agents. Starting from the CSR 2006, EFSA applied statistical methods in analysing trends at Community level. Data from MSs that have provided data consistently over the years from national control or monitoring programmes were used and the MS-specific results were weighted by their production/animal population figures when estimating Community or MS group prevalence. MSs report a substantial amount of data each year on zoonotic agents in a wide range of different animal species and food categories under the framework of the Zoonoses Directive 2003/99/EC. Therefore, there is a need to specify where the following of trends would be most important as well as the set of data needed for the trend analyses. It is important to carefully choose the fields where this would be performed. The harmonised fields should provide information that is relevant and useful at Community level and that can also be analysed in a meaningful way. The resources available for the monitoring of zoonoses and food-borne pathogens in MSs are limited and therefore the objectives of trend monitoring must be well chosen and no unnecessary burdens should be created for MSs. The data received from MSs form a complex dataset at Community level. Therefore, it is also important to assess the appropriateness of the statistical methods applied in the trend analyses. EFSA is also in the process of reviewing statistical analyses of trends in order to evaluate the most appropriate methods to be applied. In this report the following terms are used to describe the following of trends for zoonotic agents in food and animals and the different types: trend monitoring in this report means collecting data on the occurrence of zoonoses, zoonotic agents and observing the differences over the years. This term is used to describe generally the activities in observing and analysing trends over years; 5

Trend monitoring is divided in this report into the following categories: trend watching means passive monitoring of data for possible trends; and trend analyses means the statistical analysis and epidemiological interpretation of occurrence/prevalence data in order to detect significant changes over time. This report aims to give general guidance on the monitoring of trends over time in the data reported annually by MSs on the occurrence of zoonoses and zoonotic agents in animals and food at Community and MS group level. This includes examples for the estimation of sample sizes needed to detect trends. Trends in the occurrence of antimicrobial resistance are not covered by this report. 2. Current data collection and trend analyses 2.1. Current data collection system Since 2005, data on zoonoses reported in accordance with the Zoonoses Directive have been submitted to the European Commission through a web-based reporting system, and stored in a large database, hosted and maintained by EFSA. The web-based reporting system allows reporting officers in MSs to enter data concerning zoonotic agents in foodstuffs, animals and feedingstuffs collected through a variety of different systems, i.e. official monitoring and control programmes, Hazard Analysis and Critical Control Points (HACCP) and own-check programmes, and surveys. The data are entered into the database by use of a number of predefined tables, pick lists for variables, and text templates. However, the reporting officer may add new lines, footnotes to the tables as well as text forms if relevant. Based on public health priorities 5,6,7 the Zoonoses Directive lists eight zoonoses and zoonotic agents to be monitored, and data are collected on a mandatory basis on the following zoonotic agents: Salmonella, thermophilic Campylobacter, Listeria monocytogenes, verotoxigenic E. coli (VTEC) Mycobacterium bovis, Brucella, Trichinella and Echinococcus. Furthermore, the mandatory reported data include antimicrobial resistance in Salmonella and Campylobacter isolates, food-borne outbreaks and susceptible animal populations. Also, based on the epidemiological situations in MSs, data have been reported on the following zoonotic agents: Yersinia, Lyssavirus (the causative agent of rabies), Toxoplasma, Cysticerci, Sarcocystis, Coxiella burnetti (the causative agent of Q fever), Chlamydophila psittaci (formerly known as Chlamydia psittaci) (the causative agent of psittacosis) and Leptospira spp. Data on antimicrobial resistance in the indicator E. coli and Enterococci isolates are also reported. Recently (since 2006), MSs have also submitted data concerning other microbiological contaminants such as histamine, staphylococcal enterotoxins and Enterobacter sakazakii (Cronobacter spp.), for which food safety criteria are set down in Community Regulation (EC) 1441/2007 8. In 2007, data were submitted by all 27 MSs and two non-mss (Norway and Switzerland). 5 Opinion of the Scientific Committee on Veterinary Measures relating to Public Health on Food-borne Zoonoses (adopted 12 April 2000), http://ec.europa.eu/food/fs/sc/scv/out32_en.pdf. 6 Opinion of the Economic and Social Committee on the Proposal for a directive of the European Parliament and of the Council on the monitoring of zoonoses and zoonotic agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC, and the Proposal for a regulation of the European Parliament and of the Council on the control of Salmonella and other food-borne zoonotic agents and amending Council Directives 64/432/EEC, 72/462/EEC and 90/539/EEC. OJ C 94, 18.4.2002, p. 18. 7 Opinion of the Economic and Social Committee on the Resistance to antibiotics as a threat to public health OJ C 407, 28.12.1998 p 7. 8 OJ L 322, 7.12.2007, p. 12. 6

Decision 2119/98/EC 9 on setting up a network for the epidemiological surveillance and control of communicable diseases in the Community, as complemented by Decision 2000/96/EC 10 on the diseases to be progressively covered by the network, established the data collection on human communicable diseases from the MSs. Since 2005, ECDC has provided data on zoonotic infections in humans, as well as their analyses for the CSR. Until now, data used for analysis were derived from several disease networks: the European Surveillance System (TESSy), implemented and maintained by ECDC, and two Dedicated Surveillance Networks: Enter-Net and Euro-TB. This report will not address the following of trends in zoonoses cases in humans. When the annual data have been submitted by the MSs (deadline 31 May each year), data are collated, analysed and presented in the CSR. The data flow for the CSR is shown in Figure 1. Member States Animal, food and feed monitoring Foodborne outbreaks Communicable human diseases EuroTB ZCC DTU: Technical University of Denmark; ZCC EFSA s Zoonoses Collaborating Centre Figure 1. Scheme of the data flow for the CSR, 2006-2007 2.2. Data characteristics EFSA s database on zoonoses contains the most comprehensive collection of data on zoonotic agents and antimicrobial resistance in food and animals in the European Union. Since 2005, MSs have allocated substantial resources for reporting information from an extensive number of samples, detailed descriptions of monitoring systems and general evaluations of the national situation to the web database. Currently, 27 MSs and two non-mss have reported varying amounts of information for 9 OJ L 268, 3.10.1998, p.1. 10 OJ L 28, 3.2.2000, p. 50. 7

the period 2004 to 2007. Providing a common database for all MSs has improved the quality and comparability of data reported for the CSR and has helped to identify data available for the analysis of trends. The introduction of the web-based reporting application with its reporting tables and pick lists has harmonised the format of the reporting and standardised the categories of agents, animals, foods and feeds used. Results from the substantial numbers of units tested are reported each year by MSs. The total numbers reported have been the highest in the case of tests for parasites carried out in meat inspections, where 135 million tested units were reported. From the other agents, Salmonella had over 350,000 food units and 170,000 animal units tested by MSs and in the case of Listeria, Campylobacter and VTEC the total numbers of units tested varied between 20,000 and 50,000. Animal data The data received on zoonotic agents in animals tend to be more harmonised than that of foodstuffs. MSs have put in place specific control or eradication programmes required by Community legislation. Certain control and surveillance activities for zoonoses or zoonotic agents in MSs are already harmonised by European Community legislation. The degree of harmonisation varies according to related legislation. The fields harmonised by the Community legislation include the: control and reporting of brucellosis and tuberculosis in cattle, sheep, goats and pigs (Council Directives 64/432/EC 11, 91/68/EEC 12, Decision 2003/886/EC 13 ) control of certain Salmonella serotypes in poultry flocks for which specific reduction targets have been established (Regulation (EC) No 2160/2003 14 ); monitoring of antimicrobial resistance in Salmonella isolates from animals (Decision 2007/407/EC 15 ); and the control of Trichinella and certain other zoonotic parasites in the context of meat inspection (Regulation (EC) No 2075/2005 16 ). MSs may also have some additional national control or monitoring programmes for other zoonotic agents in animals. Furthermore, meat inspections provide a substantial amount of harmonised data on the occurrence of parasites in slaughter animals. Regulation (EC) No 2160/2003 on the control of Salmonella and other specified food-borne zoonotic agents, foresees the setting of Community targets in the reduction of Salmonella prevalence in poultry and pigs. So far, the targets have been set for breeding flocks of Gallus gallus (Regulation (EC) No 1003/2005 17 ), laying hens of Gallus gallus (Regulation (EC) No 1168/2006 18 ), flocks of broilers (Regulation (EC) No 646/2007 19 ) and flocks of turkeys (Regulation (EC) No 584/2008 20 ). MSs have established national control programmes to meet the targets set and these control programmes have 11 OJ 121, 29.7.1964, p. 1977. 12 OJ L 46, 19.2.1991, p. 19. 13 OJ L 332, 19.12.2003, p. 53. 14 OJ L 325, 12.12.2003, p. 1. 15 OJ L 153, 14.6.2007, p. 26. 16 OJ L 338, 22.12.2005, p. 60. 17 OJ L 170, 1.7.2005, p. 12. 18 OJ L 211, 1.8.2006, p. 4. 19 OJ L 151, 13.6.2007, p. 21. 20 OJ L 162, 21.6.2008, p. 3. 8

been approved by the EC. These control programmes vary to a certain extent but the objectives to be achieved are uniform. In addition, EFSA has issued recommendations for the harmonisation of the monitoring and reporting of antimicrobial resistance in commensal Escherichia coli and Enterococcus spp. isolates from food animals (EFSA, 2008). Food data The main part of the data regarding the occurrence of zoonotic agents in food in MSs derive from official controls undertaken by the competent authorities and the related monitoring of food by food business operators in the context of their own-checks and HACCP systems. Most MSs have some type of centralised national coordination of sampling, such as annual control and monitoring plans that define with varying details what food items should be sampled and analysed and sometimes also the number of samples taken. Regulation (EC) No 882/2004 21 on official controls that have been in application since 1 January 2006 and the relative guidance document (EC, 2006), obliges MSs to have a multi-annual plan for official controls of food, feed and animal health, and this will improve further the planning and coordination of sampling. Some MSs apply, regularly or occasionally, specific surveys to examine the agents in foodstuffs. These surveys are often well designed and limited in duration and provide data of good quality. Community legislation also lays down a number of microbiological criteria for foodstuffs, which has guided the surveillance and sampling performed in MSs (Regulation (EC) No 2073/2005 22 and Regulation (EC) No 1441/2007) e.g. by defining reference methods and the food categories of interest. 2.3. Current trend watching and analysis In the CSRs for the years 2005-2007, the data received on zoonotic agents from the MSs have been generally examined for the existence of possible trends over the years (trend watching). This has been the case when data of good quality that are relatively comparable, have been available from several years. In particular, trend watching has been carried out for data on the occurrence of zoonotic agents, such as Salmonella and VTEC, in different food categories (proportions of positive units out of the tested ones) and for data on the prevalence of certain zoonoses, such as rabies and echinococcosis in animals. Also the proportions of food units in non-compliance with the Community microbiological criteria in different years have been examined for possible trends. Furthermore, in the field of antimicrobial resistance in isolates from animal and food origin, the data have been regularly checked for possible indications of trend, i.e. in proportions of resistant isolates to certain antimicrobial substance over the years. This type of trend watching has proven to be useful in the analyses of developments in MSs and the EU, even though the data have to be carefully interpreted to account for the underlying uncertainties and possible false indications of trends. In the CSR 2006, statistical trend analyses were conducted for the first time. Areas of high public health risk, where sufficient data were available, were selected for analysis, and only MSs with three years of data (more than 25 samples) were included. 21 OJ L 165, 30.4.2004, p. 1. 22 OJ L 338, 22.12.2005, p. 1. 9

Trend analyses (statistical analyses) in CSRs 2006 and 2007 were carried out for: 1. Salmonella in: fresh broiler meat (S. spp.) (pooled sampling stages); table eggs (S. spp.) (only in 2006); breeding flocks of Gallus gallus: egg production line and meat production line (S. spp. and S. Enteritidis + S. Typhimurium); flocks of laying hens (S. spp., S. Enteritidis + S. Typhimurium); and flocks of broilers (S. spp., S. Enteritidis + S. Typhimurium). 2. Thermophilic Campylobacter in: fresh broiler meat (pooled sampling stages); flocks of broilers. 3. Bovine tuberculosis and bovine/caprine/ovine brucellosis in: infected/positive herds of cattle, sheep and goats in Community co-financed non-free MSs. 4. Echinococcus in: cattle and sheep/goats at slaughter (only in 2006). Data that were used for the statistical analysis of temporal trends in the CSRs for 2006 and 2007 consisted of the number of tested units, the number of positive units, and total number of units - population size - in each MS, for three years (2004-2006) in the CSR 2006; and for four years (2004-2007) in the CSR 2007. Graphical visualisation was carried out by trellis graphs, where the positive proportion of zoonotic agents during each year was plotted for each MS. Confidence intervals (CIs) were also plotted. In Figure 2, the percentage of Salmonella-positive samples of broiler meat, by year and MS is represented. The width of 95% CI, represented by vertical bars, is a measure of precision of the estimates. The CI is closely associated with the sample size that was tested and identifies a range of values (in this case, percentage positivity) which are likely to include the true value of the parameter in the population. Consequently, large sample sizes are associated with narrow CIs and relatively precise estimates, conversely, estimates obtained using small sample sizes will be characterised by low precision and wide CIs. In calculating CIs that are reported in Figure 2, potential non-independence of sampled units was not taken into account, and simple random sampling was assumed. Therefore, the reason for including such CIs was to provide information on the sample size and the consequent precision of each point estimate. Figure 3 presents the Salmonella-positive samples of broiler meat by year at Community level (data from 11 MSs). Community level or MS group level means are weighted means that were estimated by weighting the MS-specific proportion of positive units with the reciprocal of the sample fraction, e.g. weighted by the total number of units per MS per year, divided by the number of tested units in the MS per year. Appendix A describes more in detail the statistical methods used in the trend analyses. 10

2004 2005 2006 2004 2005 2006 20 Austria Belgium Estonia Finland 15 10 Percent of positive samples 5 0 20 15 Germany Greece Ireland Slovenia Sweden UK Italy 20 15 10 5 0 10 Year Figure 2. Plot of percentage positivity for Salmonella spp. in broiler meat samples in 11 MSs, from 2004 to 2006. Vertical bars represent 95% exact binomial CIs Weighted percent of positive samples 0 2 4 6 8 10 5 0 2004 2005 2006 2004 2005 2006 2004 2005 2006 Year Figure 3. Weighted percentage of broiler meat samples that were positive for Salmonella spp. for each year from 2004 to 2006. Data from 11 countries were weighted by the reciprocals of the sampling fraction 11

3. Rationale for monitoring the trends and criteria for selection of the fields where to monitor trends There are a number of reasons for monitoring trends over years in the occurrence of zoonoses and zoonotic agents, both at Community level and within individual MSs. First and foremost, monitoring of trends provide legislators, risk managers and other stakeholders with information on developments in public health and food safety, which would allow the consideration of needs for any intervention. The monitoring of trends also offers tools to evaluate the impact of control and eradication programmes. Furthermore, it is also important to make the most of the data being collected, and thereby optimise the use of resources. Trends over time may be monitored at EU level (i.e. in all or most MSs), at MS group level (when data are available only from a limited number of MSs) and at MS-specific level. The guidance given in this report covers only the monitoring of trends at Community or MS group level. An EU trend could be considered if data from the majority of MSs, for example from 16 MSs and 75% of relevant population, are available. In the Community, the main part of food safety and animal health legislation is harmonised, and there is a common market for food and animals within the EU. While managing this Community legislation the Commission and MSs need to follow the situation and developments in food safety in the entire Community. Therefore, monitoring trends at Community level is of particular relevance for those diseases and infections, which the Community aims to eradicate or combat through control or eradication programmes (e.g. brucellosis and tuberculosis and Salmonella in animals). Trends are also important for determining whether the Community microbiological criteria (e.g. for Listeria and Salmonella) or targets for prevalence (e.g. for Salmonella) are met and if these legislations have improved the food safety situation. In addition, monitoring trends helps to identify areas with increasing prevalence/incidence of a zoonotic agent as well as antimicrobial resistance that could potentially affect the whole Community. Furthermore, monitoring trends at MS group level helps to identify different types of developments within the Community. This makes it possible to identify which regions of the Community control efforts should receive the main focus and also to facilitate a better understanding of the overall situation across the whole Community. Interpretation of trends requires sufficient background information describing MS-specific sampling procedures and control programmes, in order to evaluate, for example, whether an observed increasing trend is a result of an actual increasing prevalence or the result of intensified sampling, investigations or reporting. Therefore, it is essential for trend monitoring that sufficient data be available (number of MSs, number of years, number of samples) and of good and comparable quality. 3.1. Types of trend monitoring The monitoring of trends in zoonotic diseases or zoonotic pathogens in certain food categories or animal populations can be divided into two types: trend watching and trend analysis; the following definitions are used in this report: trend monitoring in this report means collecting data on the occurrence of zoonoses, zoonotic agents and observing the differences between the years. This term is used to describe generally the activities in observing and analysing trends over years; 12

Trend monitoring is divided in this report into the following categories: trend watching means passive monitoring of data for possible trends; and trend analyses means the statistical analysis and epidemiological interpretation of the occurrence/prevalence data in order to detect relevant changes over time. The difference between trend watching and trend analyses is in the strength of evidence to support the existence of trend over years. In trend analyses statistical methods are used to test the probability that the trend exists, while in trend watching no such methods are applied. Trend watching is therefore giving indications where temporal trends may occur without making statements if the apparent trend is a real one or not. The benefit of trend watching is that it can be used for many types of data, whereas trend analyses set much stricter requirements for the data to be used in the analyses. In many cases, trend watching can be regarded as the first and preliminary step in trend monitoring, and in case indications for trends are observed in trend watching, the possible trends could, whenever possible, be tested with trend analyses. When trend analyses would not be possible due to poor data quality, attempts to get better data to clarify the situation can be made. Trend watching may be applied to the examination of data derived both from harmonised and nonharmonised monitoring that were reported by MSs for the Community annual zoonoses report. Trend analysis is applied to the examination of data that is more harmonised between the years and MSs. A trend analysis may be warranted or triggered if, e.g. an unexpected important increase or an emerging phenomenon is observed through trend watching or more detailed data on possible trends are needed. A trend analysis may also be conducted for certain zoonoses or zoonotic agents based on the predefined selection criteria. These selection criteria are discussed hereafter. 3.2. Criteria to select fields where to analyse trends Certain general criteria can be applied when selecting areas for trend analyses at MS group and at Community level. The zoonotic diseases/infections to be covered by trend analyses should be relevant from the public health perspective and there could be reasons to expect changes taking place in the occurrence of the agent. However, to enable meaningful trends analyses, it is also essential that sufficient comparable data be available, e.g. number of years/number of MSs reporting/number of tested units per year per MS. Generally, a minimum of three years of data points in time are needed to underpin meaningful trend analyses. Typically, there is a need for trend analysis when, based upon intervention, substantial changes in prevalence of a zoonotic agent or incidence of a zoonosis can be expected or have to be realised. Data for trend analysis are preferably collected by harmonised programmes. This need should be based on one or more criteria. The chosen criteria are applicable for a longer time period if general public health priorities do not change. Therefore, it is important to notice that the application of the same criteria may result in different zoonotic agent/ foodstuff or animal population combinations at different times due to the developments in the incidence and prevalence of agents in humans, animals and food. For the application of the criteria, information is needed from several sources, such as surveillance of human diseases, estimation of the burden of the diseases in humans, food-borne outbreaks, monitoring of agents in foodstuffs and animals in MSs, from research activities as well as from published scientific literature. 13

The criteria that could be used when selecting the fields where to analyse trends at EU or MS group level are as follows: Category A: Criteria for the selection of an agent 1. When the reported number of human cases of the zoonosis is substantial (at least 10,000 cases) in the EU, and most MSs report human cases. 2. When the disease in humans is severe to such an extent that even lower numbers of reported cases warrant trend analysis. 3. When changes in the numbers of reported human cases have been observed over several years, which would require a more in-depth follow-up. 4. When there has been an observed increase in the occurrence of the zoonotic agent in animal reservoirs or in food or when there are important observed changes in the agent, its virulence, antimicrobial resistance, host range, or reservoirs. 5. When there are hypotheses suggesting that changes should be expected in the future in the occurrence in humans, animals or food, e.g. when Community legislation lays down specific harmonised control measures/criteria or targets or control programmes for the zoonotic agent. Category B: Criteria for the selection of the food and animal category 1. When the foodstuff category or animal population is considered or perceived to be one of the main sources or important reservoir/vehicle of human infections. 2. When Community legislation lays down harmonised control measures/criteria or targets for the specific food category or animal population. 3. Regarding food, when the chosen foodstuff is consumed in most MSs. 4. When there is not a more appropriate stage in the food chain to follow the trend for the chosen agent/food or animal population combination. Category C: Conditions and other considerations 1. When the trend analyses would be important for both the Commission and MSs. 2. When there are sufficient harmonised data available in MSs on the agent in the food/animal population. 3. When there are available control measures and intervention options to reduce the occurrence of the agents in the chosen population. 4. When the occurrence of the agent is at a level high enough (in most cases >1%) to enable and justify trend analyses. 14

Trend analysis is foreseen in cases where following results are achieved by application of the criteria: Criteria A: the reply has to be positive to question 1 and/or question 2 and then a positive reply to one of the questions 3, 4 or 5; Criteria B: the reply has to be positive to question 1 and/or question 2 and then positive replies to questions 3 and 4 are needed; Criteria C: the reply has to be positive for the questions 1, 2 and 4. 4. Fields where to analyse trends A number of zoonotic agent/foodstuff or animal population combinations were evaluated based on the above criteria and the outcome of this application is presented in Appendix B. Combinations that were selected suitable for trend analyses at Community or MS group level are listed in Table 1. The evaluation was made based on the data available for the years 2004-2007. It should be noted that the proposed list is dynamic and should always be based on the latest scientific literature and Community legislation currently in force. The list should be revised on a regular basis. Furthermore, it is advisable to bear in mind that in the case of zoonotic agent/foodstuff or animal population combinations not appearing in Table 1, general trend watching can always be carried out. In the case of data on antimicrobial resistance in zoonotic agents, it was decided that at this stage it would not be appropriate to perform statistical trend analyses due to difficulties to extrapolate these results to the relevant population. The appropriate epidemiological and statistical analyses for this purpose still need to be developed further. Therefore, the need to conduct trend analyses for the occurrence of antimicrobial resistance in zoonotic agents derived from animals and food was not evaluated in this report. The issue of the annual sample size (number of samples taken) is addressed in the following chapter. In the case of mandatory Salmonella control programmes and tuberculosis and brucellosis eradication programmes, the yearly sample sizes are automatically so high that the trend analysis is feasible. For the other zoonotic agent/ foodstuff or animal population combinations, meaningful trend analysis can most likely be applied when data from several years or time points are available. 15

Table 1. Zoonotic agent/ foodstuff or animal population where trend analyses are suitable at EU level or MS group level Agent Food or animal category Harmonisation 1. Salmonella Fresh broiler meat (Salmonella spp.) 2. Salmonella Fresh pig meat (Salmonella spp.) 3. Salmonella 4. Salmonella Flocks of laying hens (Salmonella spp., S. Enteritidis, S. Typhimurium ) (approved control programme) Flocks of broilers (Salmonella spp., S. Enteritidis, S. Typhimurium) (approved control programme) 5. Salmonella Slaughter pigs (Salmonella spp.) 6. Salmonella Breeding pigs (Salmonella spp.) 7. Salmonella 8. Thermophilic Campylobacter 9. Listeria monocytogenes 10. Mycobacterium bovis 11. Brucella 12. Brucella 13. Verotoxinogenic Escherichia coli (VTEC) O157 14. Echinococcus multilocularis 1 Table 2 defines the scenarios Flocks of fattening turkeys (Salmonella spp., S. Enteritidis, S. Typhimurium) (approved control programme) Fresh broiler meat Smoked fish Infected/positive herds in cofinanced non-free MSs (approved control and eradication programme) Bovine infected/positive herds in co-financed non-free MSs (approved control and eradication programme) Caprine/ovine infected/positive herds in co-financed non-free MSs (approved control and eradication programme) Cattle hide (at the slaughterhouse) Red fox (at MS group level) Sample size Scenario (No) 1 Needed, Decision 2007/516/EC available 23 2 Needed, Commission Decision 2006/668/EC 24 2 available Regulation 1168/2006 2 Regulation 646/2007 2 Will be issued based on Regulation 2160/2003 Will be issued based on Regulation 2160/2003 Regulation 584/2008 2 Needed, Decision 2007/516/EC available Needed, Decision 2010/75/EU 25 available Directive 64/432/EEC 3 Directive 64/432/EEC 3 Directive 91/68/EEC 3 EFSA specifications issued EFSA specifications under preparation 2 1 1 3 2 2 23 OJ L 190, 21.7.2007, p. 25. 24 OJ L 275, 6.10.2006, p. 51. 25 OJ L37, 10.2.2010, p.55. 16

5. Statistical aspects of trend analyses minimum required sample size An important statistical aspect related to the trend analysis of the occurrence of zoonotic agents in food or animal populations is the justification of the minimum required sample size to be taken at different time points enabling the detection of a specified trend over time. The minimum sample size in this context means the minimum number of sampling units that should be taken at different time points from the target population to enable meaningful trend analyses. It should be noted that sample sizes for detecting specified trends over time are not appropriate as such to estimate, with a specified accuracy, the (baseline) prevalence of the agent at a specific time point. This is because trend analysis aims at detecting the change in prevalence over time not at estimating the baseline prevalence at a specific time point. A general introduction to sample size calculations for estimating the prevalence at a specified time point and for testing for differences between prevalence between two time points, as well as for testing for trends over time is presented in Appendix C. In the context of the annual CSR on zoonoses, the data submitted annually can be viewed as a series of surveys on different subjects, and terms such as monitoring surveys or repeated surveys could be used. The following sections give guidance on the MS-specific sample sizes that enable the reliable detection of specified trends at the EU level and/or at the level of a group of MSs. It should be kept in mind that the usefulness of monitoring data for trend analysis depends on the underlying (routine) data collection and monitoring scheme, notably whether or not the units tested have been randomly selected from the target population, and on the sample size used. As regards the sample sizes needed in the annual monitoring/surveys, it should be noted that the sample size is defined by two factors: the accuracy of the (baseline) prevalence estimated wanted and, on the other hand the possibility of detecting trends. In most cases, an accurate prevalence estimate is wanted on each sampling time point (year), and therefore the sample size determination is mostly dominated by this. Then secondly, if the trends are to be analysed, this could be taken into account, for example by evaluating, after how many time points (years), the yearly sample size would enable reliable trend analyses. 5.1. Periodic sample size at Member State level enabling trend detection at EU level This section includes examples of minimum required sample sizes, at MS level, to enable the detection of trends over time, at EU level and/or at the level of MS group. In this context, trend detection means the detection of changes in prevalence in a target population over time, for example via the conduct of repeated monitoring surveys. The statistical significance of trends is evaluated via regression analysis, i.e. of the annually submitted aggregated MS data by the investigation of the statistical significance of the slope of the resulting regression line. These examples are not intended to be used as sampling schemes as such, since the appropriate sample size needed for monitoring schemes is determined both on the basis of the desired baseline prevalence estimate accuracy to be obtained, as well as the magnitude of trend to be detected and the number of time points (years) after which a trend can be observed. Thus, these examples include typically only part of the information to be accounted for. The minimum sample size means the minimum number of samples to be taken by every MS, at every time point in the case of the CSR meaning mostly every year - enabling the detection of an EU level or a MS group level trend by using aggregated annual zoonoses data. It should be noted that in the case of census sampling (when every flock or herd under the programme is sampled), e.g. as is the case for Salmonella, brucellosis and tuberculosis control and eradication programmes, these minimum sample sizes are automatically satisfied. 17

Determination of the minimum required sample sizes needed to detect a trend of a certain magnitude was made through simulations in this report. The assumptions made and the mathematical and simulation methods used are presented in Appendix D. Four different scenarios were hypothesised each with a specific combination of a starting prevalence and trend (at EU level or at MS group level a change in prevalence over time) that should be detected (Table 2). Table 2. Definitions of scenarios with different starting prevalence and trend at EU level or MS group level to be detected used for simulation-based sample size determination Starting EU prevalence Trend (change per time point) at EU level or MS group level Scenario 1 50% (high) -10% (decrease) Scenario 2 10% (moderate) -10% (decrease) Scenario 3 2% (low) -10% (decrease) Scenario 4 2% (low) +10% (increase) The simulation results for the defined scenarios are presented in three-dimensional graphs (Figures 4-7) and Table 3 summarises the simulation results for each scenario. The required sample sizes resulting from the simulations are rounded to the closest upper multiple of five. Each sampling design is sized so that the EU level (or MS group level) trend can be detected with a probability of 95% (i.e. a 5% probability of missing the existing trend). The figures indicate the probability of missing a trend for different combinations of minimum required sample sizes and number of time points. In general, a trend is observable with lower sample sizes when the number of monitored time points (e.g. years) after the baseline time point gets bigger. If the probability (risk) of missing a trend is considered acceptable at a level of 5% or less, the following conclusions can be drawn: When the starting prevalence in the target population is high (50%), a decreasing trend of 10% per time point can be observed with a minimum sample size of 245 taken twice (at two time points) after the baseline time point or with a minimum sample size of 65 taken three times (at three time points) after the baseline time point. In the former case, a minimum total of 490 (= 2 x 245/year) samples must be taken by every MS and in the latter case a minimum total of 195 (= 3x 65/year) (Figure 4 and Table 3) to detect a trend at EU level or at the level of a group of MSs. In the case of a moderate starting prevalence (10%) decreasing by 10% per time point, more samples are required, and if every year a minimum of 220 samples are collected by every MS, the EU trend would be detected within four time points after the baseline time point, i.e. with a minimum total of 880 (= 4 x 220/year) samples taken by every MS (Figure 5 and Table 3). In the case of a low starting prevalence (2%), a substantially higher number of samples are needed to detect reliably a decreasing trend of 10% up till four time points after the baseline time point. After five or six time points, reasonable sample sizes, respectively 615 and 375 per year, would enable the detection of the trend (Figures 6, and Table 3). In the case of five time points the minimum total MS-specific sample is 3,075 (= 5 x 615/year), whereas in the case of six time points the minimum total MS specific sample is 2,250 (= 6 x 375/year). For the observation of an increasing trend of 10% with low starting prevalence (2%), a lower number of samples is required (Figure 7 and Table 3) compared to the observation of a decreasing trend starting at the same low starting prevalence (2%) (Figure 6 and Table 3). 18