Zoonoses and zoonotic agents in humans, food, animals and feed in the Netherlands 2001

Zoonoses and zoonotic agents in humans, food, animals and feed in the Netherlands 2001 onderzoek in dienst van mens en milieu RIJKSINSTITUUT VOOR VOLKSGEZONDHEID EN MILIEU

Zoonoses and zoonotic agents in humans, food, animals and feed in the Netherlands 2001 Zoonoses and zoonotic agents in humans, food, animals and feed 1

Edited by W. van Pelt W.van.Pelt@RIVM.nl S.M. Valkenburgh Stasja.Valkenburgh@KvW.nl Working group J.H.M. Nieuwenhuijs Jan.Nieuwenhuijs@KvW.nl A.M. Henken Andre.Henken@RIVM.nl R.E. Komijn R.E.Komijn@RVV.agro.nl F. van Knapen F.vanknapen@VVDO.vet.uu.nl Editorial group Chapter 1 Laboratory based surveillance + Frame 1 W. van Pelt W.van.Pelt@RIVM.nl W.J.B. Wannet Wim.Wannet@RIVM.nl Epidemiological research + Frame 2 Y.T.H.P. van Duynhoven Y.van.Duynhoven@RIVM.nl M. de Wit Matty.de.Wit@RIVM.nl Surveillance and monitoring of zoonotic agents J.H.M. Nieuwenhuijs J.H.M.Nieuwenhuijs@KvW.nl Integrated monitoring of zoonotic bacteria in farm animals: A.W. van de Giessen Arjen.van.de.Giessen@RIVM.nl N. Voogt Nelly.Voogt@RIVM.nl G. Visser Gerard.Visser@KvW.nl M. Bouwknegt Martijn.Bouwknegt@RIVM.nl W.D.C. Dam-Deisz Cecile.Deisz@RIVM.nl Frame 3: Monitoring of primates in Zoos W. Schaftenaar W.Schaftenaar@RotterdamZoo.nl Frame 4: Control Programmes in poultry I. Stoelhorst I.Stoelhorst@pve.agro.nl Surveillance of food products E. de Boer Enne.de.Boer@KvW.nl H. van der Zee Henk.van.der.Zee@KvW.nl Chapter 2 Salmonella B.R. Berends B.R.Berends@VVDO.vet.uu.nl W. van Pelt W.van.Pelt@RIVM.nl Campylobacter A. Havelaar Arie.Havelaar@RIVM.nl J.A. Wagenaar J.A.Wagenaar@id.wag-ur.nl B. Duim B.Duim@id.wag-ur.nl W.F. Jacobs-Reitsma W.F.Jacobs-Reitsma@id.wag-ur.nl R.J.L. Willems Rob.Willems@RIVM.nl Verocytotoxin-producing E. coli Y.T.H.P. van Duynhoven Y.van.Duynhoven@RIVM.nl R.D. Reinders R.D.Reinders@VVDO.vet.uu.nl W.J.B. Wannet Wim.Wannet@RIVM.nl A.E. Heuvelink Annet.Heuvelink@KvW.nl Mycobacterium R.E. Komijn R.E.Komijn@RVV.agro.nl D. van Soolingen D.vanSoolingen@RIVM.nl Norwalk-like Caliciviruses M. Koopmans Marion.Koopmans@RIVM.nl Trichinella J.W.B. van der Giessen Joke.van.der.Giessen@RIVM.nl Yersinia Y.T.H.P. van Duynhoven Y.van.Duynhoven@RIVM.nl E. de Boer Enne.de.Boer@KvW.nl Listeria H. van der Zee Henk.van.der.Zee@KvW.nl Brucella R.E. Komijn R.E.Komijn@RVV.agro.nl F. van Zijderveld F.vanZijderveld@ID.Wag-ur.nl Chapter 3 Rabies W. van der Poel Wim.van.der.Poel@RIVM.nl G. Visser Gerard.Visser@KvW.nl Bartonella L.M. Schouls LM.Schouls@RIVM.nl J.F.P. Schellekens J.Schellekens@RIVM.nl Borrelia, Ehrlichia and Babesia L.M. Schouls LM.Schouls@RIVM.nl J.F.P. Schellekens J.Schellekens@RIVM.nl F. Jongejan F.Jongejan@vet.uu.nl Hantavirus J. Groen Groen@viro.fgg.eur.nl Viruses in primates J. Groen Groen@viro.fgg.eur.nl Echinococcus J.W.B. van der Giessen Joke.van.der.Giessen@RIVM.nl Toxocara P. Overgaauw P.Overgaauw@wxs.nl Animal influenza virus infection A.D.M.E. Osterhaus et al. Osterhaus@viro.fgg.eur.nl Chapter 4 Monitoring of antimicrobial resistance D.J. Mevius D.J.Mevius@id.dlo.nl W.J.B. Wannet Wim.Wannet@RIVM.nl A. van de Giessen Arjen.van.de.Giessen@RIVM.nl Vancomycin-resistant enterococci R.J.L. Willems Rob.Willems@RIVM.nl Antibiotic resistance in food animals and associated public health risks A.E. van den Bogaard A.vandenBogaard@CPV.Unimaas.nl E.E. Stobberingh Est@lmib.azm.nl R.J.L. Willems Rob.Willems@RIVM.nl 2 Inspectorate for Health Protection and Veterinary Public Health

Preface This report on zoonoses presents a summary of the occurrence of zoonotic agents in human, food, animals and feed in the Netherlands. The report is based on data compiled according to the zoonoses directive, 92/117/EEC, supplemented by data obtained from Dutch surveillance and control programmes and from relevant research projects by the different institutions that have contributed to the preparation of this report. This report also gives an overview of the surveillance systems of humans and animals and who is involved and/or responsible for running the programmes. It also includes information about antibiotic resistance surveillance in food animal species, the relation with animal owners and the possible origin of animal vancomycin-resistant enterococci found in humans? It does not replace the national report because the results are not documented by year. The report will be updated if necessary, but not each year, and will be available on the web site of the Inspectorate for Health Protection and Veterinary Public Health: www.keuringsdienstvanwaren.nl. References are available by the authors. Abreveations of institutes and organisations involved: AID General Inspectorate EUR Erasmus University Rotterdam GD Animal Health Service GGD Municipal Health Service ID-Lelystad Institute for Animal Science and Health IGZ Inspectorate for Health Care KDD Animal Feed Sector Inspection Service LCI National Coordination Centre for Infectious Diseases LNV Ministry of Agriculture, Nature Management and Fisheries PDV Product Board for Animal Feed PVE Production Boards for Livestock, Meat and Eggs RIVM National Institute for Public Health and the Environment RVV National Inspection Service for Livestock and Meat UM University of Maastricht VVDO Department of the Science of Food of Animal Origin VWS Ministry of Public Health, Welfare and Sport W&V/KvW Inspectorate for Health Protection and Veterinary Public Health Zoonoses and zoonotic agents in humans, food, animals and feed 3

Contents Introduction 7 Demographic data 7 Notifiable diseases 8 Number of animals slaughtered in 2000 8 Chapter 1 - Surveillance and Monitoring 9 1.1 Surveillance and epidemiological studies of zoonotic gastroenteritis in humans Laboratory-based surveillance 9 Epidemiological research 9 Frame 1: Early warning for infections with zoonotic enteric pathogens in humans 10 Frame 2: Case-control component in the NIVEL GP sentinel and the SENSOR population study 13 1.2 Surveillance and monitoring of zoonotic agents 14 1.3 Integrated monitoring of zoonotic bacteria in farm animals 14 Frame 3: Monitoring of primates in zoos 16 Frame 4: Control programmes for Salmonella and Campylobacter in poultry 17 1.4 Surveillance of zoonotic agents in food products 19 1.5 Surveillance of zoonotic agents in feed 19 Chapter 2 - Food-Borne Zoonoses 20 2.1 Salmonella 20 2.2 Campylobacter 28 2.3 Verocytotoxin-producing Escherichia coli (VTEC) 32 2.4 Mycobacterium 33 2.5 Norwalk-like caliciviruses 35 2.6 Trichinella 36 2.7 Yersinia enterocolitica 37 2.8 Listeria monocytogenes 38 2.9 Brucella 39 4 Inspectorate for Health Protection and Veterinary Public Health

Chapter 3 - Direct and Environment-Mediated Zoonoses 40 3.1 Rabies virus 40 3.2 Bartonella henselae 41 3.3 Borrelia, Ehrlichia and Babesia 41 3.4 Hantavirus 42 3.5 Viruses in primates 43 3.6 Echinococcus 43 3.7 Toxocara 44 3.8 Influenza 45 Chapter 4 - Antimicrobial Resistance 47 4.1 Monitoring of antimicrobial resistance 47 4.2 Vancomycin-resistant enterococci (VRE) 52 4.3 Antibiotic resistance in food animals and associated public health risks 55 Zoonoses and zoonotic agents in humans, food, animals and feed 5

6 Inspectorate for Health Protection and Veterinary Public Health

Introduction Zoonoses are diseases that are transferable between animals and humans. The Ministry of Public Health, Welfare and Sport (VWS) and the Ministry of Agriculture, Nature Management and Fisheries (LNV) are both responsible for monitoring and control of these zoonotic diseases. VWS is responsible for the monitoring and control of zoonoses in the human population. It makes decisions on acceptable levels of contamination in food or prevalence of the disease in animals. The Inspectorate for Health Protection and Veterinary Public Health (W&V/KvW) falls under the ministry and is responsible for advising the minister about the status of public health in relation to food-borne and animal zoonoses. W&V/KvW have there own laboratories for research, in the field of animal zoonotic diseases. And may use facilities at the National Institute for Public Health and the Environment (RIVM). In addition, the laboratory of virology at the Erasmus University Rotterdam EUR) is important for the study of viral animal diseases. The Inspectorate for Health Care (IGZ), also a part of VWS, is responsible for monitoring and surveillance of zoonotic diseases in humans. The RIVM coordinates most of these programmes. Notifiable human zoonotic diseases are notified to the Municipal Health Service (GGD) and registered by the IGZ. At the local level, the GGD is responsible for action to minimize the public health implications of a zoonotic disease. If more than one GGD is involved the National Coordination Centre for Infectious Diseases (LCI) coordinates all actions and research to identify the source of an infection. LNV is responsible for setting up plans to minimize the risk to humans of infection by zoonotic diseases of animals. The Institute of Animal Science and Health (ID-Lelystad) carries out most of the relevant research. The National Inspection Service for Livestock and Meat (RVV) is involved in meat inspection and in the control of animal diseases. Notifiable animal diseases are notified to the local district officer and registered by the Central Office of the RVV. At the request of LNV, the Animal Health Service (GD) takes official samples in animal disease control programmes. The Commodity Boards for Livestock, Meat and Eggs conducts the control programme of Directive 92/117/EC under the responsibility of LNV. A zoonosis can be transmitted from animals to humans in various ways. Foodstuffs of animal origin are the most important source of zoonoses in humans. Salmonella and Campylobacter are the major agents for foodborne zoonoses in the Netherlands. This report includes a chapter about antimicrobial resistance that reports the results of recent research into the antibiotic resistance of microorganisms in humans and farm animals. An important purpose of this monitoring programme is the detection of potential public health risks. Antimicrobial resistance is becoming an increasingly important issue in the prevention and control of human and animal zoonoses in the Netherlands. Demographic data Dutch population in January 2000 Age distribution Total 0-4 years 983,491 5-14 years 1,962,052 15-24 years 1,883,351 25-44 years 5,019,959 45-64 years 3,862,655 > 65 years 2,152,442 Total 15,863,950 Number of animals (x 1000) and farms registered in 2000 Animals Farms Cattle 4,040 45,820 Pigs 13,118 14,524 Sheep 1,308 17,592 Goats 179 3,801 Poultry 104,014 3,860 broilers 50,937 1,094 layers 44,036 2,076 Zoönoses Zoonoses in Nederland zoonotic en Europa, agents 2001 in humans, food, animals and feed 7

Notifiable diseases Zoonosis IZW GWWD Anthrax Botulism - Brucellosis BSE - Campylobacteriosis - Echinococcosis - EHEC/VTEC - Leptospirosis (L. hardjo) Listeriosis - Psittacosis Rabies Salmonellosis - Toxoplasmosis - Trichinellosis Tuberculosis Yersiniosis - Number of animals slaughtered in 2000 Animal species (x1000) Total Cattle (incl. veal calves) 2,247 Pigs 18,583 Horses and ponies 4 Sheep 471 Goat 17 Ducks and turkeys 8,875 Poultry 483,090 broilers 463,415 hens 19,675 IZW Infectious Diseases Act (human) GWWD Animal Health and Welfare Act (animals) 8 Inspectorate for Health Protection and Veterinary Public Health

Chapter 1 Surveillance and Monitoring 1.1 Surveillance and epidemiological studies of zoonotic gastroenteritis in humans Laboratory-based surveillance, Frame 1 Laboratory Surveillance Infectious diseases The sentinel-based surveillance programme on bacterial pathogens, called Laboratory Surveillance Infectious diseases (LSI), has been running since 1989 and involves 15 regional public health laboratories (PHL), covering 62% of the Dutch population. Each first isolate of Salmonella (16 PHLs, coverage 64%), E. coli O157 (coverage 38% in 1999) and others, including Yersinia until 1996, obtained by routine diagnostic requests must be reported using a standard form. Basic information on the patient is collected, such as age, gender, residence, country of infection and possible source of infection. On a weekly basis, these laboratories also report the total numbers of observed pathogens, including Campylobacter, and the total number of stool samples examined. All first isolates of Salmonella are sent to the RIVM for serotyping, phagetyping and testing for sensitivity to antibiotics. Isolates of E. coli O157 have been sent for confirmation and further typing since 1996. Enhanced laboratory surveillance for VTEC O157 For the enhanced surveillance all Dutch medical microbiology laboratories were requested to report positive results of VTEC O157 to the municipal health services. They were also asked to send in the isolate to the RIVM for confirmation and further typing. Except for O- en H-typing, isolates are typed using verocytotoxin genotyping (VT1 and VT2) and pulsed field gel electrophoresis. Isolates were also assessed for the presence of the E. coli attaching and effacing gene (eae-gene) and the enterohemolysin gene. From April 1999, the municipal health services were requested to interview all diagnosed cases using a standardized questionnaire on risk factors and clinical aspects. Epidemiological research, Frame 2 was performed between 1996 and 1999. The study population encompasses all sentinel practices of the Dutch Sentinel Practice Network (NIVEL) and is representative for the Dutch population, according to age, sex, geographical area and urbanization, and includes 1% of the Dutch population. Samples and completed questionnaires were sent to the RIVM. The samples were tested for Campylobacter, Salmonella, Yersinia, Shigella by culture; E. coli O157/VTEC by culture and PCR; rotavirus group A, adenovirus 40/41 and astrovirus by ELISA; Norwalk- and Sapporo-like viruses by PCR, Giardia lamblia, Dientamoeba fragilis, Endolimax nana, Cryptosporidium and Cyclospora by microscopy on fixed samples and some non-pathogenic parasites. Population-based epidemiological research (Sensor 1999) Although the study in general practices (see above) has the advantage of including high numbers of more severe gastroenteritis cases, it also has the limitation that it cannot provide insight into the total incidence of gastroenteritis in the general population in the Netherlands. A population-based cohort study is the most appropriate research method to gain insight into the total incidence, microbiology and burden of gastroenteritis in the Netherlands. The most recent population study, called the Sensor study (1999), has just been finalized. Two consecutive groups of individuals were recruited at random, although stratified by age group, from the practice population of participating NIVEL general practices. These two groups were followed up for a six-month period. The case definition is comparable with the one used in the general practice case-control study, with one additional criterion. However, data were collected in such a manner that cases based on this additional criterion in the population study can be excluded to allow comparison with the sentinel study in general practices. Stool samples of cases and matched controls (case-control component, see Frame 2) were analysed for the same panel of pathogens as in the NIVEL GP study, with the addition of Bacillus cereus and bacterial toxins for Clostridium perfringens and Staphylococcus aureus. General-practice-based research (NIVEL 1996-1999) In the Netherlands, sentinel studies in general practices provide insight into the burden of the health care system and in the incidence, agents and risk factors for gastroenteritis in a population presenting to general practitioners. In these studies a sample of general practitioners is asked to report every patient consulting with gastrointestinal complaints according to a well-defined case definition (enumeration part). In addition, the relevant patients (and a matched control) are asked to fill out a questionnaire and to collect a stool sample for microbiological evaluation (case-control component, see Frame 2). The most recent sentinel study Zoönoses Zoonoses in Nederland zoonotic en Europa, agents 2001 in humans, food, animals and feed 9

Frame 1 Early warning for infections with zoonotic enteric pathogens in humans One of the tasks of the surveillance of infectious diseases is the early detection of outbreaks of infections to allow timely interventions. Automated national, interregional and regional systems for the detection of outbreaks of laboratory-confirmed infections in humans have proved to be of value in several countries. Since April 1998 an algorithm has been implemented for Salmonella in an application at the National Reference Laboratory (NRL) at the RIVM. The Dutch NRL is a reference laboratory for Salmonella spp. isolated from both human and non-human sources. From human patients all first isolates of Salmonella, collected from 16 regional public health laboratories, are sero- and phage typed. This covers 64% of the Dutch population. In September 1998 Campylobacter spp., E. coli O157 and Rotavirus were added, for weekly aggregated data only. From trends in historical data, the algorithm computes a projected frequency of occurrence for each Salmonella type and a tolerance level for the actual frequency above which a potential outbreak is indicated (Figure 1.1.1). Automated evaluation is especially helpful for monitoring a large number of different microorganisms: for Salmonella alone 600 types have already been identified since 1984 in humans in the Netherlands and an additional 400 from non-human sources. The algorithm has to be as sensitive as possible but should minimize the number of false alarms, taking into account seasonal fluctuations, secular trends and down-weighting past outbreaks in the estimations to achieve this. Retrospectively, 48 (CI 95 25-70) Salmonella types were seen on average each week, whereas 2 (P 99 8) exceeded the tolerance level for a median outbreak period of three weeks (CI 95 14-70 days). Investigation of recorded outbreaks showed that infections caught within the Netherlands were noted about 2 to 3 weeks after the onset of the disease and infections contracted abroad after 3 to 4 weeks. The outbreak warning application is an add-on to an extensive information system on Salmonella in which historical data on earlier outbreaks, regional distribution of cases and trends in occurrence in sources from both human, (farm) (exotic) animals, foods and the environment can be inspected. The same holds with respect to the development of antibiotic resistance. Together these facilities are the first step in the process of verifying signals of outbreaks. Apart from detection of manifest outbreaks in humans, the system provides indications of emerging infections or developments of resistance to antibiotics in animal husbandry that may pose a threat to human health ( Figure 1.1.2 and 1.1.3). Internet The whole information system is updated almost each week and available on the Internet within the RIVM. The Internet site can be reached from outside the RIVM using a password that is regularly changed. The password is available for the human and veterinary inspections, the food inspection service, animal health service, and, in principal, to animal production boards and co-workers in the Netherlands and abroad. Results are regularly reported in the Infectieziekten Bulletin (http://www.isis.rivm.nl/inf_bul/home_bul.html) Figure 1.1.1 Example showing an explosion of infections with S. Brandenburg at the end of 1999 caused by ox-sausage 12 10 Salmonella Brandenburg 21/11-26/12 1999 Almere, Haarlemmermeer region 10 Frequency, 16 PHL's 8 6 4 2 0 07/01/96 25/02/96 14/04/96 02/06/96 21/07/96 08/09/96 27/10/96 15/12/96 02/02/97 23/03/97 11/05/97 29/06/97 17/08/97 05/10/97 week (ending at sunday) Observed Expected Tolerance 23/11/97 11/01/98 01/03/98 19/04/98 07/06/98 26/07/98 13/09/98 01/11/98 20/12/98 07/02/99 28/03/99 16/05/99 04/07/99 22/08/99 10/10/99 28/11/99 16/01/00 Inspectorate for Health Protection and Veterinary Public Health

Figure 1.1.2 Example that illustrates the emergence of S. Paratyphi B var. Java in poultry meat, which appears to be of negligible impact on humans so far (compare Table 2.1.5) 33 Salmonella Paratyphi B var Java 0,4 30 27 24 0,35 0,3 21 0,25 % of isolates per reservoir 18 15 12 9 0,2 0,15 0,1 % of isolates in humans 6 3 0,05 0 1994 1995 1996 1997 1998 1999 2000 0 Pig(%) Cattle(%) Chicken(%) Chicken retail(%) Human(%) Zoönoses Zoonoses in Nederland zoonotic en Europa, agents 2001 in humans, food, animals and feed 11

Figure 1.1.3 Example that illustrates the emergence since 1990 of multiresistant S. Typhimurium DT104, in pigs, cattle and humans (upper figure). It apparently stabilises in humans in 2000. The early warning application (lower figure) shows a continuous increase since October 2000 stabilising at 4x normal, indicating a new increase in humans in 2001 20 Salmonella Typhimurium ft401 + ft506 (Dt104) 18 16 14 % of isolates per reservoir 12 10 8 6 4 2 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 Pig(%) Cattle(%) Chicken(%) Human(%) 15 14 13 12 16-6-96 Region: diffuse Salmonella Typhimurium ft506 (DT104) 3-10-99 Region: diffuse 26/11/2000-20/5/2001 Region: diffuse 11 10 9 8-9-96 Region: diffuse 12 Frequency 16 PHL's 8 7 6 5 4 3 2 1 0 7-Jan 10-Mar 12-May 14-Jul 15-Sep 17-Nov 19-Jan 23-Mar 25-May 27-Jul 28-Sep 30-Nov 1-Feb 5-Apr 7-Jun 9-Aug 11-Oct 13-Dec week (ending at sunday) Observed Expected Tolerance 14-Feb 18-Apr 20-Jun 22-Aug 24-Oct 26-Dec 27-Feb 30-Apr 2-Jul 3-Sep 5-Nov 7-Jan 11-Mar 13-May Inspectorate for Health Protection and Veterinary Public Health

Frame 2 Case-control component in the NIVEL GP sentinel and the Sensor population study A selection of the NIVEL sentinel practices participated in a case-control study, which was conducted between May 1996 and May 1999. The general practitioners invited every patient consulting for gastroenteritis to participate in the study as a case. For every case, the next patient consulting for complaints other than gastroenteritis in the same age group (0-11 years, 12 years and older) was invited to participate in the study as a control. Cases and controls received a similar questionnaire and a stool sample kit. In the Sensor population study, all cases reporting gastroenteritis were recruited for the nested case-control study and an age-sex matched control was selected from the population cohort. Controls were asked to collect two stool samples for a period of two weeks, cases were asked to collect four samples for a period of four weeks. Self-administered questionnaires were used to collect information on personal characteristics; long- and short-term risk factors and health-related quality of life. Cases also kept a medical diary in the four weeks following the onset of symptoms. The figure below shows some of the results of the case-control study in the GP sentinel study. Note that for comparison of cases and controls percentages are not corrected for deviations of the age/sex distribution from the general population as for Campylobacter and Salmonella in Chapter 2. Furthermore, note that cases for Yersinia all concerned non-pathogenic types and that the positive findings of VTEC were all non-o157 cases, apart from 1 O157 case (see also section 2.3). Clearly, Salmonella and Campylobacter are rarely observed in asymptomatic controls, more so for Rotavirus and NLV (Norwalk-like virus) and almost equally so for Giardia. Hence, the classical bacterial agents are the most clear cause for visiting a GP for gastroenteritis, but viruses explain a large number of the gastroenteritis diagnoses as well. In the population study, however, it is the other way around; viruses prove to be the predominant cause of gastroenteritis in the general population (see also section 2.5). Case-control study nested in GP sentinel study on gastroenteritis 11 10 9 8 Positive cases (%) 7 6 5 4 3 2 1 0 Salmonella Campylobacter Yersinia VTEC Rotavirus Norwalk-like virus Sapporo-like virus Cryptosporidium Giardia cases controls Zoönoses Zoonoses in Nederland zoonotic en Europa, agents 2001 in humans, food, animals and feed 13

1.2 Surveillance and monitoring of zoonotic agents in animals Surveillance of zoonotic agents in farm animals is necessary to gain an indication of prevalence in animals and of seasonal and annual fluctuations for use in the analysis of risk factors for human and animal populations. Control programmes are already available for some zoonotic animal diseases, such as Mycobacterium bovis in animals, brucellosis in cattle and sheep, salmonellosis in poultry, trichinellosis in pigs, wild boars and horses and rabies in animals, including bats. These control programmes are laid down in European Commission directives. The Netherlands is officially free from bovine tuberculosis (Decision 99/476/EC and 99/466/EC). Rules for trade in cattle with regard to tuberculosis and brucellosis are laid down in Directive 64/432/EEC, amended by D.97/12/EC and D.98/46/EC. For tuberculosis the methods of control for maintaining the official tuberculosis-free status are the obligation to notify animals suspected or diagnosed positive for bovine tuberculosis, the official post-mortem examination of all bovine animals slaughtered, and post mortem examination of dead animals sent to the Animal Health Service. In addition, tuberculin testing is carried out in connection with the accreditation of cattle sperm centres (Directive 88/407/EC), tracing and neighbourhood screening of cases of bovine tuberculosis and export to non-member states. The methods of control for maintaining the official brucellosis-free status are: the obligation to notify animals suspected or diagnosed positive for bovine brucellosis; notification of cases of abortion by sending blood samples of the dam within 7 days of abortion to a laboratory designated by the official veterinarian; a surveillance programme of all animals older than 24 months in 20% of the existing cattle herds by serological tests on bulk milk or individual blood samples; and bacteriological examination of aborted foetuses. Serological testing is also carried out in connection with the accreditation of cattle sperm centres (Directive 88/407/EC) and on animals exported to non-member states. If there is an outbreak of brucellosis, contact herds are traced and neighbouring herds screened. For trichinellosis the testing of fresh meat from pigs and horses is carried out by the RVV in accordance with Appendix 1 of Directive 77/96/EEC. Pooled samples from pigs (maximum 100 animals) and pooled samples from horses (maximum 10 animals) are tested using the digestion method (detection limit: 1 larva/gram). Suspect cases of rabies (Directive 92/65/EC) are sent to the Animal Health Institute, ID-Lelystad for examination. In exceptional cases, other institutes, the RIVM and the Erasmus University Rotterdam, are also involved, for instance in the research for the responsible agent for rabies in fruiteating bats in a zoo. For salmonellosis, Directive 92/117/EEC is the basis for the control programme for breeding flocks and is included in the action plans for broilers and layers. These plans are described in Frame 4. The Animal Health Service (GD) conducts the investigations in the reproduction sector. The Production Boards for Livestock, Meat and Eggs (PVE) monitors Salmonella and Campylobacter in poultry. The monitoring programmes are based on the action plans in the poultry industry for broilers, layers and turkeys. The National Inspection Service for Livestock and Meat (RVV) and the Animal Feed Sector Inspection Service (KDD) monitor the presence of Salmonella in feed. They have been designated by the Ministry of Agriculture, Nature Management and Fisheries (LNV) and the Product Board Animal Feed (PDV) respectively for monitoring compliance with the legal provisions governing trade in animal proteins and the production of and trade in animal feeds. Compliance with the Ministry of LNV and PDV regulations is monitored. Monitoring of compliance with the GMP regulations is also provided for in a PDV regulation. The prosecution of offenders is the responsibility of the General Inspectorate (AID) of the Ministry of LNV. In 1997, the RIVM started a surveillance programme on zoonotic agents in farm animals on behalf of the Inspectorate for Health Protection and Veterinary Public Health (KvW) (see also Frame 2a). The Salmonella and Campylobacter isolates are also tested for antibiotic resistance. Most of the surveillance of zoonotic diseases in pet animals, such as cat scratch disease, and in the environment, such as investigations of ticks for infectious agents or Trichinae in wild boar and foxes, is performed by the RIVM and the Veterinary Faculty in Utrecht. The laboratory of Virology of the EUR and the RIVM are involved in research into viral zoonoses,. The EUR is important for research into haemorrhagic diseases, influenza in animals and human, viruses in zoo animals and import zoonoses in humans, and the RIVM for gastroenteritis viruses. 1.3 Integrated monitoring of zoonotic bacteria in farm animals Certain zoonotic bacteria, especially Salmonella spp., Campylobacter spp. and Escherichia coli O157, are recognized worldwide as major causes of gastroenteritis (GE) in humans. The source of these infections often has its origin in the animal population, mainly in farm animals. For instance, S. Enteritidis infections have predominantly been associated with consumption of eggs or egg-containing foods (Giessen et al. 1999). Poultry is considered to be the most important source of Campylobacter infections in the Netherlands (Giessen 1996) and cattle are main reservoir for E. coli O157 (Heuvelink et al. 1998). Adequate control of these zoonotic bacteria, and thus human GE, depend on reliable data on the prevalences and trends of the agents in farm animals and foods of animal origin and on their relevance for human infections. Therefore, the Inspectorate of Public Health commissioned the National Institute of Public Health and the Environment (RIVM) to initiate an integrated monitoring programme in the Netherlands for the bacteria mentioned above, which started in 1997. Faecal samples of laying hens, broilers, veal calves, fattening pigs and dairy cattle are collected weekly on farms throughout the Netherlands. The Animal Health Service in the Netherlands performs the selection of farms in all sectors except the veal farms. The latter are supplied by the Foundation for Quality Guarantee of Veal (SKV). Participation of farms in the monitoring programme is voluntary. Calculation of the sample size is based on statistical principles with a flock as the subject of analysis (Table 1.3.1). 14 Inspectorate for Health Protection and Veterinary Public Health

The term flock describes all animals of similar age kept in one building for laying hens, broilers and veal calves. For dairy cattle, the term flock (i.e. herd) indicates all lactating and dry animals; for pigs it indicates all animals housed within one building. When calculating sample sizes, stratification is applied for the region in which a farm is located. Sector-specific stratification is applied to dairy farms, pig fatteners and veal farms for farm size and to veal farms for age as well. Sampling is performed in one flock per farm; if several flocks are present, one is randomly selected by the sampler. According to the size of the flock, a number of faecal samples, with a maximum of 60 per flock, is taken from the floor or the manure conveyor. The number of samples to be taken is based on a 95% confidence level and a 5% detection limit. These samples are subsequently pooled to form a maximum of five pooled samples per flock, with a maximum of 12 faecal samples per pooled sample. These pooled samples are sent to the RIVM for bacteriological screening for Salmonella spp., Campylobacter spp. and E. coli O157 within 48 hours of sampling. Thereafter, the samples are stored at 70 C and released for screening for zoonotic parasites and viruses in other projects. Just after or prior to sampling, a questionnaire on farm characteristics is completed in cooperation with the farm manager. These data can be used to identify factors associated with the presence of the bacteria on a farm. This information can then be used to develop effective intervention strategies for reducing the prevalence at the farm level. Table 1.3.1 Calculated sample size per sector for 2001, based on the estimated prevalence and the given accuracy, with a 90% confidence level Laying hens Broilers Fattening pigs Dairy cows Veal calves Total number of flocks per year 1 3,114 11,816 77,370 29,467 17,310 Estimated prevalence for 2001 2 20% s 25% c 30% s 10% e 20% e Accuracy 5% 5% 5% 4% 5% Sample size 165 203 228 153 174 1 Based on data from Statistics Netherlands (CBS), 2000. 2 Based on data from the monitoring programme in previous years (1997 2000). s, c, e Based on the prevalence of Salmonella, Campylobacter or E. coli. Zoönoses Zoonoses in Nederland zoonotic en Europa, agents 2001 in humans, food, animals and feed 15

Frame 3 Monitoring of primates in zoos Protocol The close relationship between man and non-human primates justifies a special approach in the surveillance of zoonotic diseases in primate collections. In a joint effort between Dutch zoos and VWS (Inspectorate for Health Protection, Commodities and Veterinary Public Health), a protocol was established providing guidelines for keeping primates in zoos. Zoos are obliged to take account of potential risks from zoonoses to the zoo personnel. When primates are transferred to another zoo, it is important to collect serological data about the immune status of the animals. It is highly recommended to quarantine a new primate prior to admission of the animal to the collection. Interpretation of tests Once in the zoo collection, opportunistic serological examinations will be performed to monitor the immune status of the animals. A number of problems arise when performing and interpreting the available tests. First, most of the tests are based on assays developed for use in human medicine. For example, when an animal is found to have antibodies against Human T-cell Leukemia Virus, it can only be assumed that these antibodies were induced by a Simian T-cell Leukemia Virus infection. The same applies to the Human Immunodeficiency Virus and the Simian Immunodeficiency Virus. Whenever antibodies against these viruses are detected, the presence of the virus must be determined by using an appropriate PCR. Another important dilemma is the interpretation of the results themselves. Given the differences in species and the way the viruses behave in each species, the presence of antibodies against virus A in one species should not be interpreted in the same way as antibodies against the same virus in another species. It is very unlikely that a wild chimpanzee with antibodies against Ebola virus still harbours this virus if the animal appears to be in a healthy condition. The same antibody titre in a green monkey, however, might form a serious threat for humans in contact with this animal. When testing for hepatitis B, the absence of a reliable guideline for the interpretation of antibody-titre results means that the same guideline is used as in human medicine, involving differentiating between core and surface-related antibodies. Whether this is a valid method still has to be proven. An appropriate PCR must be used to differentiate between the many species-related hepatitis B viruses. To make it even more complicated, the absence of antibodies against herpes B in macaques is no evidence that the animal is free from this virus. Ninety percent of the animals belonging to this group of primates are natural carriers of herpes B and may periodically shed the virus, even in the absence of antibodies. If these observations are kept in mind, systematically performed serological surveys of primates in zoological collections contribute to a better understanding of the role primate viruses may play with regard to zoonotic diseases. 16 Inspectorate for Health Protection and Veterinary Public Health

Frame 4 Control programmes for Salmonella and Campylobacter in poultry The need to eradicate Salmonella and Campylobacter All links in the poultry chain will have to cooperate to make the Salmonella and Campylobacter problem manageable. When, at the end of 1996, it became known that the majority of chicken breasts were infected with Salmonella and Campylobacter, the Dutch Production Boards for Livestock, Meat and Eggs (PVE) drew up a number of action plans. The objective of these plans is to control and reduce Salmonella and Campylobacter in the poultry sector. The action plans will serve as extensions to the implementation of the EU zoonoses regulations. Control programmes were started for broilers in April 1997, layers in October 1997 and for turkeys in April 1999. Measures for all the links in the chain In the first place, all the individual links in the chain must comply with strict hygiene requirements. Poultry farmers must clean and disinfect all empty chicken houses and all companies must make efforts to detect Salmonella and Campylobacter. Where a flock is found to be infected, specific measures must be taken, depending on the type of company involved. Companies will often have to take measures to ensure extra hygiene or prevent crossinfection. All houses found to contain an infected flock must be investigated, after cleaning and disinfection, for the presence of Salmonella and Campylobacter. Hatcheries have to set up a specified farm plan in the context of the action plan. These actions are obligatory under PVE rules. Compound feed producers must also take extra measures. Sampling All the links in the chain must perform incoming and outgoing examinations to check for the presence of Salmonella and/or Campylobacter. From the time the day-old chicks arrive on the farm, samples are taken from the paper liners in the chickboxes for bacteriological examination. Farmers that keep laying hens must have the blood of their hens tested for S. Enteritidis or S. Typhimurium (S.E. / S.T.) antibodies. This is to be done in the last 3 weeks of the rearing period and again not more than 9 weeks before the hens are to be removed from the production house. Blood samples must be taken of at least 0.5% of the birds, with a minimum of 24 and a maximum of 60 laying hens per henhouse. Other poultry farmers must have samples taken from every batch of poultry to be tested for the presence of Salmonella bacteria. At broiler farms this is to be done shortly before the birds are due to be delivered to the slaughterhouse. For samples, broiler farmers can make a choice between 2x15 faeces swabs or 2 pairs of overshoes per house. When a batch is found positive, logistic slaughtering is obligatory to ensure complete separation in time and/or place from negative flocks. Broiler farmers and slaughterhouses must have flocks tested for the presence of Campylobacter twice a year. All production flocks are examined for Salmonella bacteria in the rearing period at 4, 16 and 20 weeks by bacteriological testing of 150 faeces samples or cloaca swabs per house. During the production period samples are taken of each hatch from every machine and tested for the presence of Salmonella. If S.E. or S.T. is found, the infected flocks will be eliminated Cleaning and disinfection All poultry farmers are obliged to clean and disinfect all empty chicken houses. Before putting in the next flock, the houses have to be tested by an institute approved by the PVE. The test consists of bacteria counts of pressure samples taken from all over the house. What actions will have to be taken next depends on the result of this hygiene test. If laying hens are found to be infected with S.E. or S.T., the houses in question must always be cleaned and then disinfected by a professional, PVE approved, disinfecting company. Tests must then be carried out to determine whether the house is free of Salmonella. Only if these tests are negative may a new batch be introduced. Otherwise the house must be disinfected once again. For other types of poultry this procedure applies to all Salmonellae. Control Inspectors of one of the five inspection organizations appointed by the Production Board will once or twice a year check whether poultry farms comply with the requirements. Hatcheries and slaughterhouses are checked four times a year and fines may be imposed for not following the instructions. Zoönoses Zoonoses in Nederland zoonotic en Europa, agents 2001 in humans, food, animals and feed 17

65 60 55 Campylobacter 120 110 100 Infected flocks (%) 50 45 40 35 30 25 20 15 10 5 90 80 70 60 50 40 30 20 10 0 0 Nov- Sep- Feb- Apr- Jun- Sep- Nov- Jan- Apr- Jun- Aug- Oct- Jan- Mar- May- Aug- Oct- Dec- 1997 1998 1999 2000 %positive flocks Human 44 40 Salmonella 90 80 36 32 28 24 20 16 12 8 4 70 60 50 40 30 20 10 First isolates, humans (15 PHL) 0 0 Nov- Sep- Feb- Apr- Jun- Sep- Nov- Jan- Apr- Jun- Aug- Oct- Jan- Mar- May- Aug- Oct- Dec- 1997 1998 1999 2000 %positive flocks Human 18 Inspectorate for Health Protection and Veterinary Public Health

Monitoring the progress of the control programme and relation with occurence in humans Between October 1997 and December 2000, most flocks were tested at the slaughterhouse (neck skin, caecal contents), broiler farm (faeces) and hatchery (inlay papers and fluff). On average 500 flocks weekly for Salmonella and 210 for Campylobacter. Data were obtained by the Production Boards for Livestock, Meat, Poultry and Eggs (PVE). The figures show the seasonal evolution (slightly smoothed) on a weekly basis of the occurence of human laboratory-confirmed cases of Campylobacter (coverage: 62% of the Dutch population) and Salmonella (coverage: 64%) and percentage of flocks positive in caeca. See further Chaper 2.1 and 2.2. 1.4 Surveillance of zoonotic agents in food products Since 1990, the Inspectorate for Health Protection, Commodities and Veterinary Public Health has been running a monitoring programme on Salmonella and Campylobacter in chicken products through 5 regional inspectorates. The methods were altered in 1996 to improve the representativeness of the sampling (number of samples, product type, selling point and region). 1.5 Surveillance of zoonotic agents in feed Salmonella is controlled by the National Inspection Service for Livestock and Meat (RVV) and the Animal Feed Sector Inspection Service (KDD). They are designated by the Ministry of Agriculture, Nature Management and Fisheries (LNV) and the Product Board for Animal Feed (PDV) respectively for monitoring compliance with the legal provisions governing trade in animal proteins and the production of and trade in animal feeds. Compliance with LNV and PDV regulations are monitored. Monitoring of compliance with the GMP regulations is also covered by a PDV regulation. The prosecution of offenders is the responsibility of the General Inspectorate (AID) of LNV. Zoonoses and zoonotic agents in humans, food, animals and feed 19

Chapter 2 Food-Borne Zoonoses 2.1 Salmonella Humans Multiresistant S. Typhimurium DT104 is the most important Salmonella emerging between 1991 and 2000, now responsible for almost one third of all infections with S. Typhimurium in humans (compare Figure 1.1.3). General-practice based and community-based epidemiological studies In the Netherlands, two studies have been performed to estimate the incidence of gastroenteritis and the associated pathogens (see also section 1.1): a study in the community in 1999 (Sensor), and a study among cases consulting a general practitioner (GP) in the period 1996-1999. The incidence of gastroenteritis in the community-based study was 283 per 1,000 person-years, and that of Salmonella in particular, 3 per 1,000 person-years. The best estimate for the incidence of gastroenteritis for which a general practitioner was consulted was 14 per 1,000 personyears (adjusted for under-ascertainment), and that of salmonellosis, in particular, was 0.5 per 1,000 person-years. In conclusion we can say that in the Netherlands, with a population of 15.76 million, 50,000 cases of salmonellosis occur each year. 5% of all gastroenteritis cases consulted a general practitioner; no specific Salmonella-gastroenteritis consultancy percentages are available. Laboratory-based surveillance First isolates of Salmonella spp. from human cases of salmonellosis and non-human sources are sent to the National Reference Centre at the RIVM, for confirmation, further typing, and sensitivity testing to relevant antibiotics (see section 1.1). Between 1996 and 2000, Salmonella was isolated in 2.3% of faecal samples, with an average annual incidence of 23.7/100,000, i.e. >3700 laboratory confirmed cases of salmonellosis per year for the whole of the Netherlands. This is 7% of all estimated cases in the population. The number of isolates of Salmonella from human cases decreased by almost one-third (30%) between 1995 and 2000 (see Table 2.1.1). Over the years, S. Enteritidis has progressively emerged as the predominant serotype, before S. Typhimurium, but has clearly decreased since 1995. PT4 is the predominant phagetype of S. Enteritidis as in other Western countries. From 1996 to 2000 recent travel abroad is reported in 6.3% of the cases (the GP-sentinel study estimate is higher, above 10%). Compared with this overall figure the relative risk that an infection with S. Enteritidis was contracted abroad was less than 1, at 0.8, and that for S. Typhimurium even lower, at 0.3. Nevertheless, infections with these two serotypes are responsible for the majority of all infections of salmonella infected patients that reported travelling. The relative risk that an infection was contracted abroad was highest for S. Paratyphi B (9.4) followed by S. Typhi (8.5), S. Virchow (2.7) and S. Hadar (2.1), all in the hit list for the whole of the Netherlands. Infections with S. Typhi have halved since 1991. Mediterranean countries scored highest for S. Enteritidis and S. Typhimurium. Indonesia scored highest for S. Typhi followed by Morocco, India and Pakistan. From 1996 to 2000 the incidence of Salmonella spp. was highest among young children (0-4 years of age), but also 30% lower than in the period 1991 to 1995. The incidence steeply decreases with age and increases again among people 60 years and older. S. Typhimurium was/is the dominant serotype among children (0-4), and S. Enteritidis among people 15-60 years of age. At the end of May isolations of Salmonella spp. slowly increase and peak in late September, generally a few weeks later than Campylobacter spp. Between 1991and 1995, however, the Salmonella season started faster and peaked by the end of July. In contrast with Campylobacter, the seasonality of Salmonella in humans is completely independent of that found in poultry at the end of the slaughterline (see Frame 4). Between 1996 and 2000 incidence in the larger Dutch cities was significantly lower than in the rest of the country. In most cities S. Enteritidis was the dominant serotype, while S. Typhimurium dominates in the rural areas. Between 1991-1995 S. Enteritidis was less dominant in the cities and the contrast between rural and urban areas was less pronounced. Table 2.1.1 The evolution of the main Salmonella serotypes in humans reported by 16 Public Health Laboratories between 1991 and 2000 (coverage: 64% of the Dutch population) Salmonella type 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 Salmonella spp. 2833 2584 2804 2980 2975 2889 2556 2266 2128 2057 S. Typhimurium 967 957 994 736 823 1002 786 686 680 605 S. Typhimurium DT104 36 54 76 94 145 218 203 187 218 183 S. Enteritidis 978 870 1050 1472 1434 1270 1163 979 862 929 S. Enteritidis PT4 877 738 904 1132 1152 1066 965 744 584 605 S. Typhi 41 48 34 50 38 22 23 15 19 17 S. Paratyphi B 15 6 5 12 16 5 18 8 19 5 Other serotypes 832 703 721 710 664 590 566 578 548 472 20 Inspectorate for Health Protection and Veterinary Public Health

Poultry and other food animals In 1997, the National Institute for Public Health and the Environment started a nationwide surveillance programme of zoonotic agents in farm animals for, and in collaboration with, the Veterinary Public Health Inspectorate (see section 1.3 and Table 2.1.2). This integrated surveillance programme focuses on VTEC O157, Salmonella spp. and Campylobacter spp. in faecal samples of poultry layer and broiler flocks, fattening pigs, veal calves and dairy cows. Approximately 1,000 farms are sampled each year. Sampling plans are based on statistical principles. Poultry In the second half of 1997 the Dutch Product Boards for Livestock, Meat, and Eggs (PVE) implemented their Action Plan (PVA) on the control and reduction of Salmonella and Campylobacter in broilers and layers (see Frame 4). The target for Salmonella in broilers was no more than 10% positive flocks at the end of the slaughterline; the target for Salmonella in layers was no more than 5% positive flocks with S. Enteritidis (S.E.) and S. Typhimurium (S.T.). With the Ministry of Public Health, Welfare and Sports it was agreed that the targets regarding Salmonella and Campylobacter should be reached within a period of about 2 to 2.5 years for broilers (i.e., should be achieved in 1999) and 3 years for layers. Figure 2.1.1 shows that, due to the PVA the percentage of Salmonellapositive flocks has been reduced considerably. However, the targets have not been reached within the time-frame set. Within a Salmonella-positive flock not all animals are infected, therefore massive cross-contamination within flocks at or just before slaughtering is indicated as positive findings in neck-skin are twice as high as in caeca. In the case of Campylobacter where almost all animals in a positive flock are infected, findings in neck-skin and caeca are about the same (Figure 2.2.1). The percentage of positive flocks at the end of the slaughterline remained a constant cause for concern. Therefore, at the end of 1999 the PVE decided that logistic slaughtering of flocks (i.e., in space and/or time separated slaughtering of negative and positive or suspected positive flocks) would become mandatory as of that moment. More recent data are required to draw any conclusions and it is still unclear whether the new demands are always met in practice. Figure 2.1.1 Percentage of Salmonella positive flocks from hatchery to the end of the slaughterline, from the last quarter of 1997 to the fourth quarter of 2000 60 Salmonella positive flocks (%) in PVE-monitoring 50 40 Positive flocks (%) 30 20 10 0 4th 1st 2nd 3rd 4th 1st 2nd 3rd 4th 1st 2nd 3rd 4th 1997 1998 1999 2000 Quarter neck skin (slaughterhouse) Caecum (slaughterhouse) faeces (broiler farm) inlay leaflets (hatchery) fluff (hatchery) Zoonoses and zoonotic agents in humans, food, animals and feed 21