Zoonoses and zoonotic agents in humans, food, animals and ... · Zoonoses are diseases that are transferable between animals and humans. The Ministry of Public Health, Welfare and

Zoonoses and zoonotic agents in humans, food, anim

als and feed in the Netherlands, 2001

Zoonoses and zoonotic agents in humans, food,

animals and feed in the Netherlands 2001 is

published by the Inspectorate for Health

Protection and Veterary Public Health

(Keuringsdienst van Waren) in close

collaboration with the National Institute

for Public Health and Environment (RIVM).

Collaborating institutes

KvW - Inspectorate for Health Protection

and Veterinary Public Health

P.O. Box 16108

2500 BC The Hague

tel 0800-0488 or +31 (0)70 3407001

fax +31 (0)70 3407080

www.keuringsdienstvanwaren.nl

RIVM - National Institute for Public Health

and the Environment

Antonie van Leeuwenhoeklaan 9

P.O. Box 1

3720 BA Bilthoven

ID-Lelystad - Institute for Animal Science and Health

P.O. Box 65

8200 AB Lelystad

GD - Animal Health Service

P.O. Box 9

7400 AA Deventer

VVDO - Department of the Science of Food

of Animal Origin

P.O. Box 80163

3508 TD Utrecht

RVV - National Inspection Service for

Livestock and Meat

Burg. Feithplein 1

Postbus 3000

2270 JA Voorburg

EUR - Erasmus University Rotterdam

Institute of virology

P.O. Box 1738

3000 DR Rotterdam

PVE - Production Boards for Livestock,

Meat and Eggs

P.O. Box 5805

2280 HV Rijswijk

University Hospital Maastricht

Departement of Medical Microbiology

P.O. Box 5800

6202 AZ Maastricht

Zoonoses and zoonotic agents in humans, food, animalsand feed in the Netherlands2001

van mens en milieu RIJKSINSTITUUT VOOR VOLKSGEZONDHEID EN MILIEUonderzoek in dienst

1Zoonoses and zoonotic agents in humans, food, animals and feed

Zoonoses and zoonotic agents in humans, food, animals and feed in the Netherlands2001

2 Inspectorate for Health Protection and Veterinary Public Health

Mycobacterium

R.E. Komijn [email protected]

D. van Soolingen [email protected]

Norwalk-like Caliciviruses

M. Koopmans [email protected]

Trichinella

J.W.B. van der Giessen [email protected]

Yersinia

Y.T.H.P. van Duynhoven [email protected]

E. de Boer [email protected]

Listeria

H. van der Zee [email protected]

Brucella


F. van Zijderveld [email protected]

Chapter 3

Rabies

W. van der Poel [email protected]

G. Visser [email protected]

Bartonella

L.M. Schouls [email protected]

J.F.P. Schellekens [email protected]

Borrelia, Ehrlichia and Babesia

L.M. Schouls [email protected]

J.F.P. Schellekens [email protected]

F. Jongejan [email protected]

Hantavirus

J. Groen [email protected]

Viruses in primates

J. Groen [email protected]

Echinococcus

J.W.B. van der Giessen [email protected]

Toxocara

P. Overgaauw [email protected]

Animal influenza virus infection

A.D.M.E. Osterhaus et al. [email protected]

Chapter 4

Monitoring of antimicrobial resistance

D.J. Mevius [email protected]

W.J.B. Wannet [email protected]

A. van de Giessen [email protected]

Vancomycin-resistant enterococci

R.J.L. Willems [email protected]

Antibiotic resistance in food animals

and associated public health risks

A.E. van den Bogaard [email protected]

E.E. Stobberingh [email protected]


Edited by

W. van Pelt [email protected]

S.M. Valkenburgh [email protected]

Working group

J.H.M. Nieuwenhuijs [email protected]

A.M. Henken [email protected]


F. van Knapen [email protected]

Editorial group

Chapter 1

Laboratory based surveillance + Frame 1



Epidemiological research + Frame 2


M. de Wit [email protected]

Surveillance and monitoring of zoonotic agents

J.H.M. Nieuwenhuijs [email protected]

Integrated monitoring of zoonotic bacteria in farm animals:

A.W. van de Giessen [email protected]

N. Voogt [email protected]

G. Visser [email protected]

M. Bouwknegt [email protected]

W.D.C. Dam-Deisz [email protected]

Frame 3: Monitoring of primates in Zoos

W. Schaftenaar [email protected]

Frame 4: Control Programmes in poultry

I. Stoelhorst [email protected]

Surveillance of food products

E. de Boer [email protected]

H. van der Zee [email protected]

Chapter 2

Salmonella

B.R. Berends [email protected]


Campylobacter

A. Havelaar [email protected]

J.A. Wagenaar [email protected]

B. Duim [email protected]

W.F. Jacobs-Reitsma [email protected]


Verocytotoxin-producing E. coli


R.D. Reinders [email protected]


A.E. Heuvelink [email protected]


PrefaceThis 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

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

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.

4

ContentsIntroduction 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

Inspectorate for Health Protection and Veterinary Public Health

5

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


Zoönoses in Nederland en Europa, 2001 7Zoonoses and zoonotic agents in humans, food, animals and feed

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 food-

borne zoonoses in the Netherlands.

This report includes a chapter about antimicrobial resistance that reports

the results of recent research into the antibiotic resistance of micro-

organisms 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.

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 contamina-

tion 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.

Demographic data

Introduction

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

Inspectorate for Health Protection and Veterinary Public Health8

Notifiable diseases

Zoonosis IZW GWWD

Anthrax ● ●

Botulism ● -

Brucellosis ● ●

BSE - ●

Campylobacteriosis - ●

Echinococcosis - ●

EHEC/VTEC ● -

Leptospirosis ● ● (L. hardjo)

Listeriosis - ●

Psittacosis ● ●

Rabies ● ●

Salmonellosis - ●

Toxoplasmosis - ●

Trichinellosis ● ●

Tuberculosis ● ●

Yersiniosis - ●

IZW Infectious Diseases Act (human)

GWWD Animal Health and Welfare Act (animals)

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

was performed between 1996 and 1999. The study population encompas-

ses 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 popula-

tion.

Samples and completed questionnaires were sent to the RIVM. The samp-

les were tested for Campylobacter, Salmonella, Yersinia, Shigella by cul-

ture; 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-patho-

genic 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.

1.1 Surveillance and epidemiological studies ofzoonotic 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 laborato-

ries were requested to report positive results of VTEC O157 to the munici-

pal 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

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 accor-

ding 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


Chapter 1

Surveillance and Monitoring

One of the tasks of the surveillance of infectious diseases is the early detection of outbreaks of infections to allow timely interventions. Automated natio-

nal, 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 compu-

tes 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 estima-

tions to achieve this. Retrospectively, 48 (CI95 25-70) Salmonella types were seen on average each week, whereas 2 (P99 8) exceeded the tolerance level

for a median outbreak period of three weeks (CI95 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)

Frame 1

Early warning for infections with zoonotic entericpathogens in humans


Salmonella Brandenburg

21/11-26/12 1999Almere, Haarlemmermeer

region

0

2

4

6

8

10

12

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

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

week (ending at sunday)

Freq

uenc

y, 1

6 PH

L's

Observed Expected Tolerance

Figure 1.1.1 Example showing an explosion of infections with S. Brandenburg at the end of 1999 caused by ox-sausage

10

Zoönoses in Nederland en Europa, 2001Zoonoses and zoonotic agents in humans, food, animals and feed

Salmonella Paratyphi B var Java

0

3

6

9

12

15

18

21

24

27

30

33

1994 1995 1996 1997 1998 1999 2000

% o

f iso

late

s pe

r res

ervo

ir

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

% o

f iso

late

s in

hum

ans

Pig(%) Cattle(%) Chicken(%) Chicken retail(%) Human(%)

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)

11


Salmonella Typhimurium ft506 (DT104)

16-6-96Region: diffuse



26/11/2000-20/5/2001Region: diffuse

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

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-Feb5-A

pr7-Jun

9-Aug

11-Oct

13-Dec

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

week (ending at sunday)

Freq

uenc

y 16

PH

L's

Observed Expected Tolerance

Salmonella Typhimurium ft401 + ft506 (Dt104)

0

2

4

6

8

10

12

14

16

18

20

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

% o

f iso

late

s pe

r res

ervo

ir

Pig(%) Cattle(%) Chicken(%) Human(%)

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

12

Zoönoses in Nederland en Europa, 2001Zoonoses and zoonotic agents in humans, food, animals and feed

Frame 2

Case-control component in the NIVEL GP sentineland 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).

0

1

2

3

4

5

6

7

8

9

10

11

Posi

tive

case

s (%

)

cases controls

Salmonella

Campylobacter

Yersinia

VTEC

Rotavirus

Norwalk-li

ke virus

Sapporo-like vi

rus

Cryptosp

oridium

Giardia

Case-control study nested in GP sentinel study on gastroenteritis

13


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 regula-

tions is also provided for in a PDV regulation. The prosecution of offen-

ders 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 farmanimals

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 con-

sumption 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).

1.2 Surveillance and monitoring of zoonotic agents inanimals

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 tuber-

culosis 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 tuber-

culosis-free status are the obligation to notify animals suspected or

diagnosed positive for bovine tuberculosis, the official post-mortem exa-

mination 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 institu-

tes, the RIVM and the Erasmus University Rotterdam, are also involved,

for instance in the research for the responsible agent for rabies in fruit-

eating 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 repro-

duction 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.

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 year1 3,114 11,816 77,370 29,467 17,310

Estimated prevalence for 20012 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.

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.


Protocol

The close relationship between man and non-human primates justifies a special approach in the surveillance of zoonotic diseases in primate collec-

tions. 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.

Frame 3

Monitoring of primates in zoos

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 cross-

infection. 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.


Frame 4

Control programmes for Salmonella andCampylobacter in poultry


Campylobacter

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Sep-

1997

Nov- Feb-

1998

Apr- Jun- Sep- Nov- Jan-

1999

Apr- Jun- Aug- Oct- Jan-

2000

Mar- May- Aug- Oct- Dec-

Infe

cted

floc

ks (%

)

0

10

20

30

40

50

60

70

80

90

100

110

120

%positive flocks Human

Salmonella

0

4

8

12

16

20

24

28

32

36

40

44

Sep-

1997

Nov- Feb-

1998

Apr- Jun- Sep- Nov- Jan-

1999

Apr- Jun- Aug- Oct- Jan-

2000

Mar- May- Aug- Oct- Dec-

0

10

20

30

40

50

60

70

80

90

Firs

t iso

late

s, h

uman

s (1

5 PH

L)

%positive flocks Human

18


1.4 Surveillance of zoonotic agents in food productsSince 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 represen-

tativeness of the sampling (number of samples, product type, selling point

and region).

1.5 Surveillance of zoonotic agents in feedSalmonella 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) respec-

tively 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 regula-

tion. The prosecution of offenders is the responsibility of the General

Inspectorate (AID) of LNV.

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.


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).

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. Enteriti-

dis 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.

2.1 Salmonella

Humans

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 person-

years (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 sero-

type, before S. Typhimurium, but has clearly decreased since 1995. PT4 is

the predominant phagetype of S. Enteritidis as in other Western countries.

Chapter 2

Food-Borne Zoonoses

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


Poultry and other food animalsIn 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 Salmonella-

positive 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.

Salmonella positive flocks (%) in PVE-monitoring

0

10

20

30

40

50

60

4th

1997

1st

1998

2nd 3rd 4th 1st

1999

2nd 3rd 4th 1st

2000

2nd 3rd 4th

Quarter

Posi

tive

flock

s (%

)

neck skin (slaughterhouse) Caecum (slaughterhouse) faeces (broiler farm)

inlay leaflets (hatchery) fluff (hatchery)

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

22

The data provided by the industry (PVE) are roughly in agreement with

the results of the national surveillance of zoonoses in poultry (compare

Figure 2.1.1 and Table 2.1.2). Comparison of columns three/four and

five/six of Table 2.1.3 shows that the methods of isolation can influence

results considerably. When using an additional method of isolation,

that was more sensitive to S. Enteritidis in particular, considerably more

flocks scored positive for Salmonella in poultry faeces.

Because the targets regarding the percentages of broiler flocks positive

for Salmonella and Campylobacter have not been reached within the

time-frame initially agreed upon, labeling of (potentially) contaminated

poultry products will be compulsory as of August 1, 2001.

LayersOn the first of November 1997 measures for the control and reduction of

Salmonella in layers were implemented under the ‘Plan of Action’ (PVA) of

the Dutch Product Boards for Livestock, Meat and Eggs (PVE). The target

set was a reduction within 3 years to less than 5% layer flocks infected

with Salmonella Enteritidis and Salmonella Typhimurium. A period of

3 years was chosen because the duration of the laying period means the


first results could be obtained only after 15 months. The control scheme

was nearly the same as for broilers (see Frame 4).

From the beginning in 1997 the infection rate in breeding flocks was very

low. In 2000 two breeding production flocks were infected with

Salmonella Enteritidis and for the first time after 5 years one rearing flock

and two breeding production flocks were infected with Salmonella

Typhimurium. The layer flocks are divided into layer rearing and layer

production flocks. The infection rates in rearing flocks for S.E and S.T

were 0.3 % and 0.1 %; in layer production these were 11.1% and 0.3 %

respectively. The target of less than 5% infected flocks has not been

reached. The risk factors for infection of layer flocks do not depend on the

housing system, and were wet manure systems and farms with flocks of

more ages; there was a positive (less infections) relation with vaccination

against S.E. One of the major changes in the PVA is the obligation to

vaccinate new flocks if the previous flock on the farm was S.E-positive.

Table 2.1.2 Surveillance of Salmonella spp. in farm animals in the Netherlands, 1997 - 2000

Category Year positive flocks/ % positives positive flocks/ % positives

flocks tested (% S.E.) flocks tested (% S.E.)

RV medium RV + DIA/MSRV medium

Broilers 1997 16/63 25 n.d.

(random) 1998 51/192 27 61/192 32

1999 21/152 14 31/152 20

2000 14/128 11 21/128 16

Layers 1997 22/114 19; (18% S. E.) n.d.

(random) 1998 24/207 12; (63% S. E.) 49/207 24; (67% S. E.)

1999 20/168 12; (15% S. E.) 38/168 23; (45% S. E.)

2000 15/166 9; (33% S. E.) 34/166 21; (53% S. E.)

Dairy cows 1997 0/84 0

(random) 1998 7/263 3

1999 4/167 2

2000 2/156 1

Veal calves 1997 3/114 3

(random) 1998 1/148 1

1999 3/60 5

2000 1/133 1

Pigs 1998 14/41 34

(random) 1999 24/190 13

2000 29/194 15 67/194 35

Legend: RV = Rappaport-Vassiliadis medium, DIA/MSRV = Diagnostic Semisolid Salmonella Medium/Modified Semisolid Rappaport-Vassiliadis medium;

n.d. = not done, % S. E. = percentage of a serotype S. Enteritidis.

Pigs and cattleThe results of the surveillance of zoonotic agents in farm animals for

Salmonella show that Dutch veal calves are not massively colonized by

Salmonella (Table 2.1.2). The control of other food-borne pathogens in

cattle, particularly E. coli O157:H7 (see section 2.3), are thus given first

priority. The percentages in pigs as found in the monitoring programme

agree with the findings for pigs and herds as found in several research

projects of the Department of the Science of Food of Animal Origin of the

Dutch Faculty of Veterinary Medicine (VVDO) (Table 2.1.3) It is clear that

the prevalence of Salmonella in pigs and on pork is the result of an

endemic stable situation which, in fact, has been improving gradually

since the early 1970s (see also Figure 2.1.2).


Because Salmonella reduction in poultry has been given first priority,

there are, as yet, no special control programmes implemented in the pig

industry. However, currently the PVE has nearly completed its ‘Action

Plan for the control of Salmonella in pigs’, and it can thus be expected

that in the near future the percentage of Salmonella-contaminated herds,

pigs and pork will decrease as well.

Table 2.1.3 Prevalence of Salmonella in samples of slaughtered pigs and prevalence of positive serum samples

D1 = sampling day 1, D2 = sampling day 2. (source: VVDO)

Prevalence (%)

Total (A+B) Slaughterhouse A Slaughterhouse B

Sample N % D 1+2 D 1 D 2 D 1+2 D 1 D 2

Serum samplesa 183 18.0 23.6 21.7 30.0 12.8 2.9 40.0

Carcass swabs 213 1.4 1.1 2.0 0 1.7 1.4 2.0

Liver swabs 150 9.3 19.3 28.1 8.0 3.2 0 12.0

Tongue swabs 150 9.3 14.0 21.9 4.0 6.4 4.3 12.0

Rectal contents 82 25.6 29.8 37.5 20.0 16.0 n.d.b 16.0

Mesenterial lymphnodes 150 9.3 15.5 15.2 16.0 5.4 6.0 4.0

Tonsils 179 19.6 27.9 26.2 32.0 11.8 11.8 12.0

Total tested for presence of Salmonella 925 10.9 17.1 20.9 11.7 6.0 4.7 8.6

a Tested for antibodies against Salmonellab n.d. = not done

FoodIn retail premises, Salmonella is monitored by the Inspectorate for

Health Protection, Commodities and Veterinary Public Health (W&V/KvW,

see section 1.2). As can be seen from Table 2.1.4, it is mainly poultry meat

products that are found positive for Salmonella. The W&V noticed a steady

decline in the percentage of positive chicken meat from 33% in 1996 to 18%

in 1999, but this increased again in 2000 to 20%. A strong decline is seen for

S. Enteritidis in 2000, when it was present in only 1% of the isolates. On the

other hand, S. Paratyphi B var Java emerged strongly, from 3% in 1995 to

10 % in 1996 and increased again, quite spectacularly, to 33% of all isolates

in 2000. The same increase has been found for less well defined other

chicken sources (see also Frame 2). At the fourth and fifth CRL-Salmonella

workshop 1999 and 2000, NRL-Salmonella Berlin reported exactly the same

pattern of emergence, but other European countries did not.


Table 2.1.4 Surveillance of Salmonella spp. in retail premises in the Netherlands, 1996-2000.

Category Year Positive samples / % positives;

samples tested) Serotype % of positives

Meat Beef 1999 7/746 1

Pork 1999 33/533 6

Mixed pork/beef 1999 22/281 8

Poultry meat Chicken products 1996 432/1325 33 ; S. E. 12%;S. PbJ. 10%

1997 382/1314 29 ; S. E. 9% ;S. PbJ. 19%

1998 218/1077 20 ; S. E. n.d. ;S. PbJ. 11%

1999 151/859 18 ; S. E. 5% ;S. PbJ. 14%

2000 305/1454 21 ; S. E. 1% ;S. PbJ. 33%

Guinea-fowl 1999 7/35 20

Turkey 1999 14/144 10

Duck 1999 3/52 6

Dove 1999 1/21 5

Ostrich 1999 1/30 3

Pheasant (wild) 1999 0/27 0

Eggs Battery cage 1999 5/23070 0.02

Floor housing 1999 5/17480 0.03

Special hen feed 1999 4/5650 0.07

Total 1999 14/46200 0.03 ; S. En. 11%, S. Ty. 1%,

S. In. 1%, S. Br. 1%.

Legend: S. E.: Salmonella Enteritidis; S. PbJ.: S. Paratyphi B var. Java; S. Ty.: S. Typhimurium; S. In.: S. Infantis; S. Br.: S. Brandenburg

The main causes for contamination of the end products are inadequate

processing of the (positive) feed components and/or post process conta-

mination (cross- and recontamination). Corrective actions undertaken by

the feed industry include: repelletizing of the feed at higher process

temperatures; acidification of the feed with, for example, formic acid; more

intensive pest control in the silo and at the loading stations; intensified

cleaning and disinfection of the plant and processing equipment; feed-

back of results of investigations regarding Salmonella to the suppliers of

the (critical) feed components, coupled with tougher contractual demands

regarding cleaning and disinfection of the deliverers’ plant, handling and

shipping of the components; and a more intensive monitoring of the

Salmonella status of the (critical) feed components delivered at the feed

mill. Considering that in 1997 the percentage of Salmonella-positive

compound feeds for pigs and poultry was about twice as high, overall the

industry has steadily improved its performance. On a yearly basis, almost

all Salmonella-positive samples derive from 5-20% of the feed mills.

The responsible feed-mills, however, may vary from period to period.

Animal feedIn the framework of article 20 of EU Directive 90/667/EU, samples of

animal feed components and compound feeds were taken regularly

during a period of one year. This one-time monitoring scheme was under-

taken throughout Europe to gain insight into the prevalence/incidence of

contaminated animal feed components and compound feeds in the

respective membership countries. In addition, samples have been taken

in the framework of EU Directive 92/117/EU for several years now. For the

year 1999/2000 both monitoring schemes were combined. Table 2.1.5

shows the results of the investigations.

In particular, the percentage of positive Salmonella samples from

imported by-products of rape seed was high (11.9%). Also, imported

by-products of linseed, sunflower-seed and Soya beans were regularly

contaminated with Salmonella spp. (3.7%, 2.7% and 2%, respectively).

In addition, almost 1% of the samples of the end product (i.e. the

compound feed) for poultry were positive for Salmonella.


Table 2.1.5 Percentage of Salmonella positive samples of feed components and compound feeds, taken under national monitoring programme,

June 1999 and July 2000

Number of positive samples/ Percentage (%)

Total number of samples

Feed components

Barley and by-products 0/137 0

Wheat and by-products 5/708 0.7

Maize and by-products 0/447 0

Ground-nut by-products 0/1 0

Rape seed by-products 127/1072 11.9

Palm kernel and by products 0/94 0

Soya and by products 22/1118 2.0

Sunflower seed by-products 9/335 2.7

Linseed by-products 3/81 3.7

Compound feeds

Poultry feed 70/7612 0.9

Pig feed 12/2883 0.4

Cattle feed 15/3099 0.5

Total 263/17587 1.5


Table 2.1.6 Calculated contribution by pigs, cattle, chickens and eggs to the relative occurrence of human salmonellosis in

the period 1994–1998 (n=13.666)

1994-1998 Humans Pigs (%) Cattle (%) Chickens (%) Eggs (%)

(%) n=2888 n=1679 n=3526 n=730

fract. contrib. fract. contrib. fract. contrib. fract. contrib. fract.

Enteritidis 1 37.1 - - 0.6 1.1 6.6 11.3 29.8 51.3

Typhimurium other 10.1 6.2 26.5 2.9 12.3 0.7 3 0.2 0.9

Enteritidis other 5.4 - - 0.1 0.3 1.4 5.5 4 16.1

Typhimurium 506 4.6 2.3 6.8 1.7 5.2 0.2 0.7 0.3 1

Typhimurium 510 3.9 3.1 11.5 0.8 2.9 - - - -

Hadar 2.3 - - - - 2.2 12 - -

Enteritidis 12 2.2 - - - - 0.6 1.3 1.6 3.5

Virchow 2.2 0.1 0.2 0.1 0.1 1.6 3.8 0.4 1

Infantis 2 0.6 7.5 - - 0.9 11.3 0.4 4.2

Bovismorbificans 1.9 1.1 0.5 0.6 0.3 0.3 0.2 - -

Typhimurium 401 1.6 1.2 4 0.5 1.5 - - - -

Typhimurium 296 1.6 1.2 1.7 0.4 0.5 0.1 0.1 - -

Enteritidis 3 1.4 - - - - 0.2 0.7 1.2 3.4

Brandenburg 1.2 0.6 1.7 0.2 0.5 0.4 1.2 - -

Typhimurium 80 1.1 0.8 2.7 0.2 0.6 - - - -

Typhimurium 20 1 0.9 2.5 0.1 0.3 - - - -

Typhimurium 507 1 0.8 2.3 0.1 0.4 0.1 0.2 - -

Typhimurium 350 0.8 0.6 2.4 0.2 0.7 - - - -

Goldcoast 0.8 0.6 0.9 0.1 0.2 - - - -

Typhimurium 61 0.8 0.5 1.7 0.2 0.5 - - - -

Panama 0.7 0.5 4 0.1 1.1 - - - -

Typhimurium 351 0.7 0.6 2.5 0.2 0.7 - - - -

Typhimurium 60 0.7 0.6 1.8 0.1 0.3 - - - -

Agona 0.6 - - - - 0.6 4.3 - -

Livingstone 0.6 0.2 2.2 - - 0.3 3 0.2 2.1

Derby 0.5 0.4 3.5 0.1 0.5 - - - -

Braenderup 0.4 - - - - 0.1 1.6 0.3 4.7

Heidelberg 0.3 - - - - 0.3 9.5 - -

London 0.3 0.3 4.3 - - - - - -

Mbandaka 0.3 - - - - 0.2 4.4 0.1 1.9

Dublin 0.2 - - 0.2 64.2 - - - -

Indiana 0.2 - - - - 0.2 3.5 - -

ParatyphiB v Java 0.1 - - - - 0.1 3.7 - -

Other types 7.4 1.7 7.4 1.1 4.1 3.7 17.3 0.8 8.9

Other sources 3,7 - - - - - - - -

TOTAL* 100 25.2 100 10.7 100 21 100 39.4 100

(24-26) (10-11) (20-22) (38-40)

Legenda: contrib. = contribution to the human cases as a percentage; fract. = fraction of all Salmonella types found in that particular source as a

percentage; * 95% confidence interval based on bootstrap analyses on all the data (10,000 bootstrap samples).

27

salmonellosis and the annual number of cases of salmonellosis has been

obtained by combining data from population studies, general practice

sentinel studies, laboratory surveillance, hospital registrations and

mortality statistics in the Netherlands, expert opinions, and estimates

from other Western countries.

The costs per case of Salmonella in the Netherlands were thus estimated

to be between NLG 1900 and 2800 (EUR 860-1266). For the period 1994-1998

the annual number of cases of salmonellosis is estimated to be between

25,000 and 105,000 cases, amounting to an average economic costs of

NLG 70-200 million per year (EUR 31.6-90.4 million).

Between 1984 and 1998 the cocktail of Salmonella types causing

salmonellosis in humans changed considerably, whereas the number of

cases of human salmonellosis than halved. Figure 2.1.2 shows the

estimated yearly economic burden of human salmonellosis and the com-

puted contributions of certain categories of farm animals (see Table 2.1.5).

Most of the reduction in human salmonellosis in Figure 2.1.2 is due to a

reduction in the number of cases with S. Typhimurium. The main reser-

voirs of these are pigs and, to a much lesser extent, cattle. However,

in the period 1997-2000 the roughly 30% decrease in human salmonellosis

also mirrored the decrease in the prevalence of S. Enteritidis, of which

poultry is the main reservoir.

Between 1984 and 1996, the absolute numbers of human salmonellosis via

poultry remained the same, making infections via poultry relatively more

important in this period. The annual costs of human salmonellosis caused

by poultry and eggs amount to an estimated NLG 45-120 million, which is

about 60% of the total estimated economic burden of salmonellosis.

Within poultry products, the role of eggs became more important in both a

relative and absolute sense between 1984 and 1996. This was due to the

epidemic increase of S. Enteritidis until 1994.

0

15

30

45

60

75

90

105

120

135

150

165

180

195

210

1984-'88 1989-'93 1994-'98

Other Cattle Pig Egg Chicken

24,5(15,5-42,5)

33(21-57,5)

44,5(28-77)

45,5(29-79,5)

35,5(22,5-61,5)

29(18,5-51)

29(18,5-50)

38(24-66)

88(55,5-152.5) 12,5 (8-21,5)

16,5(10-28,5)

40,5(25,5-70,5)

4,5 (3-7,5)3,5 (2,5-6,5)

Mill

ion

Dut

ch g

uild

ers


Relationships between food animals and human cases of salmonellosis

Cluster-analyses of the data on the isolation of the Salmonella types found

in cattle (mainly dairy cattle and veal calves), pigs (adults and piglets),

poultry (laying and broiler flocks, chicken products) and eggs (raw mate-

rials for egg products and consumption eggs) has shown that these types

are in general host-specific. This allows for a rather straightforward

computation to establish which fraction of cases of human salmonellosis

can be attributed to which category of farm animal. This is worked out in

Table 2.1.6 for the period 1994-1998. For example, S. Typhimurium Dutch

phagetype 506 (corresponding to the multiresistant phage type DT104 in

the English phage typing system) constituted 4.6% of all isolates in

humans in the period 1994-1998, and 6.8%, 5.2%, 0.7% and 1% in pigs,

cattle, chickens and eggs, respectively. Their contribution to the total of

4,6% of cases in humans is proportional to this, i.e. 2.3% for pigs, 1.7% for

cattle, 0.2% for chickens and 0.3% for eggs, respectively.

In the past five years human salmonellosis was caused in about 25% of

cases by Salmonella types originating from pigs, almost 11% from cattle,

21% from chickens, 39% from eggs and about 4% from ‘other’ sources.

However, this does not consider the fact that up to 10% of all infections

are contracted abroad or caused by imported contaminated foods,

although this last fraction obviously consists of many products that are

derived from the same categories of farm animals. Note that in 1999 and

2000 the fraction of DT104 increased to almost 10% (see Table 2.1.1).

The costs of human salmonellosis

An estimate of the costs of human salmonellosis can be obtained by

multiplying an estimate of the average cost of a case of salmonellosis by

an estimate of the total number of Salmonella infections in the general

population. An estimate of the average costs of a case of (non-typhoid)

Figure 2.1.2 Estimated economic costs of human salmonellosis per associated food source for three five-year periods

2.2 Campylobacter

Humans

General-practice based and community-based epidemiological studies

In the Netherlands, two studies have been performed to estimate the

incidence of gastroenteritis and associated pathogens (see also Chapter 1);

a study in the community in 1999 (Sensor), and a study among cases

consulting a general practitioner in the period 1996-1999.

The incidence of gastroenteritis in the community was 283 per 1,000 person-

years. Campylobacter was isolated from 2% of cases, yielding an incidence

of Campylobacter-associated gastroenteritis in the community of 6.8 per

1,000 person-years. In 1991, a population-based surveillance study on the

incidence of acute gastroenteritis was performed, leading to an age-standar-

dized estimate of 447 episodes per 1,000 person-years. Campylobacter was

isolated from 4.5% of faecal samples. Thus, both the incidence of gastro-

enteritis and the percentage of Campylobacter-positive faecal samples were

higher in 1991. Most probably, the 1991 study overestimated the incidence of

campylobacteriosis because it was carried out in spring and early summer in

a limited region of the Netherlands and used a different case-definition.

The best estimate for the incidence of gastroenteritis for which a general

practitioner was consulted was 14 per 1,000 person-years (after adjusting for

under-ascertainment). Campylobacter was isolated from 10.5% of cases in

general practice, yielding an incidence of Campylobacter in general practice

of 1.5 per 1,000 person-years. No decreasing trend over the years was obser-

ved in the percentage attributable to Campylobacter, nor in the incidence of

gastroenteritis in the GP study between 1996 and 1999. In conclusion, we can

say that in the Netherlands, with a population of 15.76 million, 100,000 cases

of campylobacteriosis occur annually.

In general, 5% of all gastroenteritis cases consulted a general practitioner;

for Campylobacter-gastroenteritis this percentage is likely to be higher.

A similar study in 1992–1993 found an incidence of 9 GP consultations for all

gastroenteritis per 1,000 person-years and Campylobacter was isolated from

14.6% of all faecal samples. Thus, against a stable background of GP visits,

the prevalence of Campylobacter has decreased between these two studies.

Laboratory-based surveillance

Fifteen regional public health laboratories have been reporting their total

number of first isolates of Campylobacter spp. each week since April

1995. These laboratories effectively cover 62% of the Dutch population

(see Chapter 1). Two laboratories (covering more than 1 million inhabi-

tants) deliver their data electronically, supplying data on age, sex,

residence and antibiotic resistance for the period 1996–2000. Between

1996 and 2000, Campylobacter was the main pathogen isolated in faecal

samples (3.4%) with an average annual incidence of 36 per 100,000,

i.e. 5,700 laboratory-confirmed cases of campylobacteriosis per year for

the whole of the Netherlands. This is 5.3% of all cases in the population.

The number of isolates of Campylobacter decreased significantly

between 1997 and 1999 (15%). However, in 2000, it was 9% higher than in

1999. There is distinct seasonal variation in the incidence of campylo-

bacteriosis. Isolates of Campylobacter strongly increase at the end of

May and peak in early September. In contrast with Salmonella,

Campylobacter seasonality in humans parallels that found in poultry at

the end of the slaughterline (see Frame 4). However, the peak season in

humans finishes a month earlier than in poultry. In accordance with the

GP sentinel study and findings elsewhere in developed countries, the inci-

dence is highest among the youngest children (0-4 years of age) and

higher, as well as in young adults (15-29). The incidence was 2 to 3 times

lower in the age classes 5-14 and those 30 years and older. Quite a large

effect was related to the degree of urbanization, incidences in rural areas

being half of those found in urban regions.

Health burden

Infection with thermophilic Campylobacter spp. (mainly C. jejuni) usually

leads to an episode of acute gastroenteritis, which resolves within a few

days to a few weeks. Occasionally, more severe and prolonged diseases

may be induced, notably Guillain-Barré syndrome, reactive arthritis or

bacteraemia. For some patients, the disease may even be fatal. Data on

the epidemiology of illness associated with thermophilic Campylobacter

spp. in the Netherlands in the period 1990-1995 was integrated in one

public health measure, the Disability Adjusted Life Year (DALY). DALYs are

the sum of Years of Life Lost by premature mortality and Years Lived with

Disability, weighed with a factor between 0 and 1 for the severity of the

illness. There is considerable uncertainty and variability in the epidemio-

logical information underlying the estimated health burden, which is

explicitly taken into account in the analysis. The health burden of illness

associated with thermophilic Campylobacter spp. in the Dutch population

has been estimated by simulation to be 1400 DALY per year (90% confiden-

ce interval 900–2000 DALY per year). The main determinants of health

burden are acute gastroenteritis in the general population (310,000 cases,

290 DALY), gastroenteritis-related mortality (30 cases, 410 DALY) and

residual symptoms of Guillain-Barré syndrome (60 cases, 340 DALY).

Poultry and other food animalsIn November 1996, the Government and the Dutch Production Boards for

Livestock, Meat, and Eggs (PVE) agreed on the implementation of additio-

nal screening and hygiene measures in different levels of the poultry

production chain. This action plan was called, ‘Plan of Action’ (PVA) and

was aimed at reducing the prevalence of Salmonella and Campylobacter

(see Frame 4, chapter 1). Between October 1997 and December 2000,

flocks were tested for Campylobacter at the slaughterhouse (neck skin,

caecum) and broiler farm (faeces); on average 210 flocks weekly

(compare section 2.1).

As shown by Figure 2.2.1 a slight reduction seems to have been achieved

in the past three years, on average from 48% in 1998 to 35% in 2000

(compare Table 2.2.1). In contrast with Salmonella, within a Campylo-

bacter-positive flock almost all animals are infected and cross-contami-

nation does not show-up, so that positive findings in neck skin and caeca

almost coincide (Figure 2.2.1). The results for Salmonella, however,

indicate massive cross-contamination within flocks at or just before

slaughtering, as positive findings in neck skin are twice as high as in

caeca (compare Figure 2.1.1).


at the end of the fattening round. Contamination with Campylobacter

usually becomes apparent after two to three weeks and gradually

increases with time. This can be seen in Table 2.2.1 which shows the

unadjusted prevalences (random throughout the cycle) to be lower then

those estimated for the end of the cycle (36-42 days) which in turn are

close to those found in caeca at the slaughterhouse in the PVE monitoring

programme (compare figures in Frame 4 with Figure 2.2.1).

The survey will yield knowledge of the prevalence and trends of zoonotic

agents in farm animal populations. Furthermore, epidemiological

relationships between the presence of pathogens in farm animals and

potential risk factors can be determined because the general information

about the farm and its management is also collected by the programme.

In 1997, the National Institute of Public Health and the Environment

started a surveillance programme on zoonotic agents in farm animals on

behalf of and in collaboration with the Veterinary Public Health Inspecto-

rate (see section 1.3). This surveillance programme focuses on VTEC

O157, Salmonella spp. and Campylobacter spp. in faecal samples of poul-

try layer and broiler flocks, fattening pigs, veal calves and dairy cows.

Approximately 1,000 farms are sampled each year and sampling plans are

based on statistical principles. Sixty fresh random faecal samples from a

flock are collected and pooled into five pooled samples. A flock is consi-

dered positive for a certain pathogen if one or more of the pooled samples

is found positive. Samples are taken randomly during a cycle and so the

data are not directly representative for the prevalence of contamination


Campylobacter positive flocks (%) in PVE-monitoring

0

10

20

30

40

50

60

70

4th

1997

1st

1998

2nd 3rd 4th 1st

1999

2nd 3rd 4th 1st2000

2nd 3rd 4th

Quarter

Posi

tive

flock

s (%

)

neck skin (slaughterhouse) Caecum (slaughterhouse) faeces (broiler farm)

Figure 2.2.1 Percentage of Campylobacter-positive flocks at the poultry farm to the end of the slaughterline, from the last quarter of 1997 to

the fourth quarter of 2000

In Table 2.2.1 summarizing the findings of the surveillance on zoonotic

agents in farm animals, the results of the PVE-monitoring program are

added. It shows that the prevalence of Campylobacter in broilers

decreased between 1997 and 1999; however, in 2000 it increased again.

This pattern was also observed in chicken products at retail (Table 2.2.2)

and in humans.

Dairy cows, veal calves and fattening pigs are also frequently contamina-

ted with Campylobacter spp., but contaminations are rarely found at retail

(Table 2.2.2). Surveillance, therefore, was discontinued for these farm

animals in 2000 and may be repeated in the future if results from,

for example, the retail sector, indicate this.

The target percentages of broiler flocks that test positive for Salmonella

and Campylobacter set in the PVA have not been reached within the

initially agreed time-frame (see Frame 4 and section 2.1) This means that

labelling of (potentially) contaminated poultry products will be compulso-

ry from 1 August 1 2001.

FoodTable 2.2.2 summarizes the available information on the prevalence of

Campylobacter in raw foods in the Netherlands, as investigated by the

W&V/KvW. Samples were taken randomly from retail outlets; sales points

for chicken products reflected sales volumes (more samples from super-

markets).

Table 2.2.1 Surveillance of Campylobacter spp. in farm animals, the Netherlands, 1997-2000

Category Year Campylobacter spp. Percentage (%1)[%2]{%3}

positives /flocks tested

Broilers 1997 21/47 (45) [63]{—}

(random) 1998 58/189 (31) [48]{48}

1999 26/151 (17) [24]{41}

2000 31/128 (24) [34]{35}

Dairy cows 1997 n.t. (random)

1998 41/130 (32)

1999 11/167 (7)

2000 n.t. Veal calves

1997 n.t. (random)

1998 52/62 (84)

1999 35/60 (58)

2000 n.t. Fattening pigs

1997 n.t. (random)

1998 37/38 (97)

1999 86/190 (45)

2000 n.t.

n.t. = not tested; (%1) unadjusted prevalence (random sampled throughout a cycle); [%2] prevalence estimated at 7 weeks using logistic regressio

analysis; {%3} prevalence found at the slaughterhouse in the PVE monitoring programme.


The data show that chicken products are still the most frequently conta-

minated type of food. C. jejuni is the species most frequently observed in

poultry samples. In the past five years a slow decrease in prevalence of

Campylobacter has been observed, from 36% in 1996 to 24% in 1999.

However, in 2000 it increased, nullifying what seemed to have been

achieved in the preceding years.

In 1994, shellfish (oysters and mussels) were also regularly contaminated

with Campylobacter, C. lari being the most frequently isolated serotype.

In 1999, no contaminated samples were observed, probably due to better

hygiene at production sites and effective use of UV irradiation of process

water. Beef, pork, fowl other than chicken and raw vegetables were

infrequently contaminated with Campylobacter.

Multilocus sequence typing and AFLP typing of Campylobacter jejuni

Seventy-five C. jejuni strains of human (including strains from three out-

breaks), animal and environmental origin from different countries were

analysed by multilocus sequence typing (MLST) of housekeeping genes

and these results were compared with the results of AFLP fingerprinting

analysis. Thirteen to 20 different alleles were identified, resulting in 54 dif-

ferent sequence types (STs). Sequence types were grouped into sequen-

ce lineages when alleles of four or more loci were identical. Assignment

of the STs into lineages revealed that 10 STs were unique and 44 were

assigned to 8 lineages. The majority of the isolates originating from

humans and poultry belonged to the two largest ST lineages, 6 and 8.

Lineage 6 contained 45% of the human and 57% of the poultry isolates and

lineage 8 contained 25% of the human and 9% of the poultry isolates.

Table 2.2.2 Surveillance of Campylobacter spp. at the retail level, the Netherlands, 1996-2000

Category Year Campylobacter spp. positive / Percentage (%)

samples tested

Meat Beef 1999 3/738 0.4

Pork 1999 0/524 0

Mixed pork/beef 1999 4/275 2

Poultry meat Chicken products 1996 480/1325 36

1997 417/1314 32

1998 282/1076 26

1999 202/859 24

2000 444/1454 31

Turkey 1999 1/145 1

Duck (wild) 1999 3/52 6

Pheasant 1999 1/27 4

Guinea-fowl 1999 1/35 3

Shellfish Shellfish 1994 52/100 52

1999 0/97 0.0

Vegetables Vegetables (raw) 1999 3/966 0.3

This means that most (70%) of the strains recovered from humans are

grouped with a large subset (66%) of poultry isolates. Housekeeping gene

sequences show that horizontal exchange of genetic information has a

major influence on the structure and evolution of C. jejuni populations.

Resistance to antimicrobial agents

Resistance to fluoroquinolones in Campylobacter isolates from humans is

increasing and is attributed to treatment of broilers with fluoroquinolones.

In 1989 resistance percentages to fluoroquinolones of Campylobacter

from broilers was 14% and from humans was 11%; in 1994 this amounted

to 33% and 19% respectively. Between 1995 and 1999 the resistance

stabilized at 21% in humans. In 1999, 17% of the broiler flocks were

treated with a fluoroquinolone. Differences between subgroups of

patients with campylobacteriosis with respect to the level of resistance to

fluoroquinolones might, therefore, besides other reasons, to a certain

extent reflect differences in exposure to Campylobacter-contaminated

poultry meat. Between 1996 and 1999 human campylobacteriosis was

3-6 times more frequent in summer as in the winter while resistance to

fluoroquinolones was found to be 3-6 times lower in the summer. Among

humans living in rural areas the Campylobacter incidence is half that

found in urban areas, while Campylobacter resistance to fluoroquinolones

is two-thirds of that found in urban areas. Furthermore, Campylobacter

resistance to fluoroquinolones in adults 20 years and older is higher than

in children, gradually declining to two-thirds of the adult level in the

youngest children.

The tentative conclusion is that in people infected in the summer months,

in young children or in people living in rural areas, a clearly larger fraction

of the Campylobacter infections might originate from sources other than


poultry. Nothing is known, however, of the species distribution. Knowing

the level of resistance of Campylobacter in broilers from week to week in

parallel to that in humans allows the computation of the approximate

fraction of human campylobacteriosis associated with poultry. To this

end, since May 2000 almost 25% of all slaughtered broilers found positive

for Campylobacter have been monitored for species distribution and

resistance.

Conclusions

Thermophilic Campylobacter species are the most frequently identified

bacterial agents of gastroenteritis in the Netherlands. The health burden

associated with campylobacteriosis is considerable, in part due to its

association with Guillain-Barré syndrome and reactive arthritis. It is

difficult to obtain an unbiased estimate of the true incidence of campylo-

bacteriosis in the general population or those consulting general

practitioners. It is even more difficult to obtain a representative picture of

the duration, severity and impacts of campylobacteriosis in the general

population. Due to methodological difficulties it is not possible to draw

conclusions on trends from comparisons of point estimates of incidence

by cohort studies. Laboratory surveillance appears to be more useful for

trend evaluations, but identifies only a minor part of all cases. If anything,

the trend has been towards a decrease in the incidence/prevalence of

Campylobacter in humans, as well as in poultry (broilers) and poultry

products, in the Netherlands up to 1999, but this seems to have reversed

in 2000. The incidence of human campylobacteriosis is still high and

additional measures are required to obtain a further reduction. MLST and

AFLP typing are emerging as promising tools for establishing genetic

relationships between strains from different sources.

32

2.3 Verocytotoxin-producing Escherichia coli (VTEC)

IntroductionVerocytotoxin-producing Escherichia coli O157, also called enterohae-

morrhagic E. coli O157 or Shiga toxin-producing E. coli O157, was recog-

nized as an important human pathogen in 1982. In the first half of the 1990s

attention for this pathogen grew due to several large outbreaks of

diarrhoea and VTEC-related complications like haemolytic uraemic

syndrome (HUS) in Japan, Scotland and the USA.

In the Netherlands, only two small clusters of VTEC have been observed.

The first in June 1993, where four young children developed HUS and

eight household members were infected. The most likely source was a

recreational lake. The second cluster involved a farm family in April 1998.

It was assumed that the first case was infected by veal calves present on

the farm. The other people were most likely infected by person to person

transmission within the household.

Humans

Results of general-practice based and community-based epidemiologi-

cal studies

From the epidemiological studies of patients with gastroenteritis in the

community and in general practices (see section 1.1) we learned that

VTEC (especially VTEC O157) was rather uncommon in the Netherlands.

In general practices, VTEC was found in 0.5% of the consulting gastro-

enteritis cases, VTEC O157 was observed only once (0.1%). In the commu-

nity, VTEC (all non-O157) was observed in 0.3% of the cases.

Results of enhanced laboratory surveillance

The enhanced laboratory-based surveillance of VTEC O157 (see Chapter 1)

identified 36 and 43 cases in 1999 and 2000 respectively, i.e. 0.23 and 0.27

per 100,000 inhabitants. Most cases (80%) were diagnosed between July

and December (Figure 2.3.1). VTEC O157 was observed in all age classes


20001999

0

1

2

3

4

5

Jan- Mar- May- Aug- Oct- Dec- Mar- May- Jul- Oct- Dec-

Date

Repo

rted

VTE

C 01

57(range 1-84), but peaked in children aged 0-4 years (25%). For 69 patients

(87%), the isolate was confirmed as VTEC O157 and further typed.

Questionnaires on risk factors and clinical aspects (see Chapter 1) were

received for 68 cases (89%). HUS was diagnosed in at least 10 patients

(15%), from which one young case died. Twenty-six patients (38%) were

hospitalized, with a median stay of 8 days. Generally recognized risk

factors, such as consumption of undercooked or raw beef (8), raw milk (1),

unpasteurized cheese (10), contact with farm animals (14) or contact with

other symptomatic persons were reported for a total of 36 (53%) patients.

Animals

Epidemiological findings

It has been shown that more than one strain can be found simultaneously

at the same farm. As much as four different strains have been isolated at

the same dairy farm. Some strains seem to disappear within a short

period of time, whereas others can persist for at least five months.

Individual animals shed the organism usually for only a limited period of

time (range approximately 2 to 8 weeks). However, non-stop shedding of

the same VTEC O157 phagetype for at least four months has been obser-

ved. In a study carried out at a slaughterhouse (July-October 1995 and

1996), VTEC O157 was isolated from 2/397 (0.5%) veal calves and 57/540

(10.5%) adult cattle. All phagetypes and verocytotoxin types of human

isolates have been identified in cattle as well.

Results of surveillance at the farm level

Surveillance in animal husbandry at the farm level (see Chapter 1) found

VTEC O157 in dairy cattle in about 7% of the farms tested between 1997

and 2000; in veal calves it increased consistently from 3% in 1997 to 17% in

2000 (Table 2.3.1). The highest prevalence was observed between July

and December. This seasonality coincides with that of the incidence of

VTEC infections in humans (see below).

Figure 2.3.1 Number of reported VTEC O157 patients, enhanced surveillance, 1999-2000

DNA fingerprinting it has recently been proven that M. microti bacteria

can cause severe forms of tuberculosis in humans. M. avium complex

bacteria are the most frequently encountered non-M. tuberculosis

complex bacteria from humans in the Netherlands. M. Avium bacteria can

cause severe forms of tuberculosis, particularly in immunodeficient

individuals.

M. bovis is the causative agent of tuberculosis in cattle, but a wide range

of other animals is also susceptible to infections with these bacteria.

In a large number of countries reservoirs of M. bovis have been found in

wildlife species as badgers, brush-tailed possum, elk and deer.

Fortunately M. bovis infections have never been diagnosed in wildlife in

this country. Occasionally M. bovis infections have been diagnosed in

Zoo- or hobby animals. Previously, bovine tuberculosis was endemic

among cattle in the Netherlands and humans were continuously infected

by the oral route. This trend was reversed after introduction of the pasteu-

rization of milk in 1940 to prevent these infections. The control programme

of routine tuberculin testing was started after the Second World War.

In 1951, 30% of the (56,166) herds were infected, but this was already

reduced to less than 5% in 1955. In 1992 the routine tuberculin testing was

stopped in the Netherlands and now surveillance of bovine tuberculosis

mainly depends on post-mortem examinations of all slaughter animals.

HumansIn the last decades, less than 1% of the tuberculosis cases diagnosed in

the Netherlands were caused by M. bovis. The age distribution of the

patients with M. bovis infections is not even. Most cases are found among

Dutch patients 65 years and older, and fewer in the age group 45-64,

and are clearly caused by endogenous reactivation from latent infections.

M. bovis infections among Dutch patients in the age group <45 years are

highly infrequent. Most cases of M. bovis infections among foreign-born

patients are found in the age category from 15- 64, and it is conceivable

that these infections are contracted outside the Netherlands. Between

13 and 15 cases of human M. bovis infections are reported each year.

M. microti, previously described by Wells et al. in the 1930s as the ‘vole

bacillus’, is one of the last known subspecies in the Mycobacterium

tuberculosis complex. About one fifth of the voles examined in the UK

were infected with these bacteria. Later on, M. microti was also found in

cats, cows, pigs, badgers, a llama, and a ferret. Until a few years ago,

these bacteria were not associated with disease in humans. However,

with the aid of molecular tools six cases of M. microti infections in the

Netherlands, two in Germany, one in the UK, and one in Switserland have

been recognised. These observations indicate that M. microti is

occasionally capable of causing severe forms of tuberculosis in humans.

The laboratory diagnosis of M. microti infections is difficult, as these

microorganisms grow very slow in vitro under restricted conditions.

M. avium complex bacteria are frequently encountered in humans and

some of these bacteria can cause severe infections, particularly in AIDS

and other immune compromised patients. The etiology of M. avium

complex infections in humans is not fully understood. DNA fingerprinting

results indicated that 90 M. avium ssp. Avium isolates from lymph nodes of

slaughter pigs had a high degree of similarity with some of the isolates

At the farm level, VTEC O157 isolated from laying hens, broilers and

fattening pigs was low from 1997 to 1999. For this reason dairy cattle and

veal calves were the only group of farm animals surveyed systematically

in 2000.

Conclusion

So far, VTEC O157 seems to be a limited public health problem in the

Netherlands (the incidence of laboratory-confirmed cases is 0.25 per

100,000 inhabitants). Nevertheless, because of the seriousness of the

illness, this pathogen deserves continuous attention and trends in

humans and animals should be monitored. Sources other than cattle and

VTEC non-O157 should not be overlooked.

Table 2.3.1 Surveillance of VTEC in farm animals, RIVM 1997-2000

Category Year Number of positives/ (% )

total number tested

Broilers 1997 0/63 0

1998 2/186 1

1999 4/100 4

2000 n.d.

Laying hens 1997 1/114 1

1998 0/202 0

1999 0/115 0

2000 n.d.

Dairy cattle 1997 6/84 7

1998 13/267 5

1999 13/161 8

2000 14/158 9

Veal calves 1997 3/113 3

1998 8/152 5

1999 8/60 14

2000 22/133 17

Fattening pigs 1997 n.d.

1998 1/41 2

1999 0/189 0

2000 n.d.

2.4 Mycobacterium

IntroductionAlthough tuberculosis in humans derived from animals sources now plays

a minor role compared with tuberculosis caused by transmission among

humans, it is highly important to monitor these infections. Therefore,

in addition to counts for cases of Mycobacterium tuberculosis infections,

isolation of M. bovis from human or animal sources is notifiable.

The Dutch cattle population have been officially free of tuberculosis since

1992, a situation that was ratified by the decision of the European

Commission 95/138/EC. However, M. bovis infections still occasionally

occur in humans, cows and other animals. Furthermore, with the aid of


from humans. The same type of bacteria have been found in potting soil,

a mixture containing peat and compost. In contrast, all 49 M. avium ssp.

Avium strains isolated from 29 different birds exhibited identical DNA

fingerprints which were hardly ever found amongst M. avium ssp. Avium

isolates from humans. Caseous lesions cause M. avium infections are

seen in lymph glands of about 0.5% of the pigs slaughtered in the

Netherlands.

M. tuberculosis is only rarely involved in zoonotic transmission, for instan-

ce between humans and monkeys, between humans and elephants and

between human and parrots.

AnimalsUntil October 1st 1999 the surveillance system to maintain the tuberculosis

free status of the cattle population and the control of outbreaks of bovine

tuberculosis were carried out autonomously by the Animal Health Service

under regulations of the Production Board of the industry. On that date,

a change in the regulations under the Animal Health and Welfare Act

made by the Ministry of Agriculture , Nature Management and Fisheries

directly responsible for bovine tuberculosis. Control of outbreaks is now

carried out by the RVV.

The surveillance on bovine tuberculosis now depends mainly on the

official post mortem examination of all bovine animals slaughtered.

In addition tuberculin testing of cattle takes place as a part of the accredi-

tation procedure for cattle sperm centres (Directive 88/407/EC), export to

non-member states and with the tracing and screening of cases of bovine

tuberculosis.

The high speed of the slaughtering process means that small tubercles

are not observed by the meat inspectors and outbreaks are sometimes

not discovered for a long period. When positive animals are discovered

a large percentage of the animals at the farm of origin are affected

(see table 2.4.1)

In addition, tuberculosis has been detected occasionally in imported

slaughter animals.

In an attempt to detect outbreaks at an early stage 1000 herds with the

greatest number of bought and sold animals were selected and tuberculin

tested in 1996 and 1997. No positive herds were detected and the tests

were stopped. The Zoo’s in the Netherlands have a control system to

avoid tuberculosis in their animals. All animals that die are sent for post

mortem. Before arrival at the Zoo or movement to an other group, animals

are tuberculin tested and blood samples are taken. If a positive case is

diagnosed all contact animals are screened.

Human contactsOutbreaks of M. bovis and M. tuberculosis in animals are reported to the

regional Inspectorate and the Central Inspectorate for Health Protection,

Commodities and Veterinary Public Health by the RVV. All people that

have intensive contact with cattle from positive farms are advised to

undergo testing for tuberculosis and are treated when necessary.

M. avium infections

Due to the all-in all out procedures and the strict hygienic measures,

M. avium infections are very rarely seen in commercial poultry. A suspec-

ted increase in the incidence of caseous lesions in lymph nodes of

slaughter pigs prompted a large-scale investigation of slaughter pigs at

five slaughterhouses in 1996. The caseous lesions were seen in 0.5% of

the total of 158,763 pigs inspected. In a follow-up study, M. avium complex

bacteria were isolated from 219 of 402 affected submaxillary or mesente-

ric lymph nodes. Ninety-one of the isolated strains were analysed by

genotyping. Only one strain was of a type also seen in birds. In the same

period MAC isolates of 191 human patients were genotyped. Analysis

showed that 59% of the porcine isolates and 61% of the human isolated

had genotype patterns that were at least 75% similar to those of the other


Table 2.4.1 Cases of tuberculosis in Dutch herds detected at meat inspection

Year Location Type of herd Number of Number of Number of

cattle positive cattle infected contact

tuberculin testing herds

1992 1. Asperen Dairy 137 84 2

2. Sprang Capelle Young stock 89 62 -

1993 3. Hilvarenbeek Beef 61 22 -

4. Veldhoven Dairy 83 65 4

1994 5. Kerk Avezaath Trade 65 0 -

1995 6. Tilligte Dairy 206 68 8

1996 7. Woerden Dairy 59 27 -

1998 8. Nederweert Beef 46 31 1

1999 9. Agelo Dairy 146 102 9

In addition to outbreaks, NLVs cause numerous sporadic cases of

gastroenteritis (11% of all cases, almost half a million community cases

annually). The risk factors for these infections are currently under investi-

gation in the Netherlands. It has been established that many different

types of NLV cocirculate in the general population, causing sporadic

cases and outbreaks.

NLV or SLV infections can be diagnosed by visualization of virus particles

by electron microscopy and with molecular methods (RT-PCR). However,

in most countries these methods are not available for routine diagnostics.

Stool viruses can be typed by sequence analysis or by reverse line blot-

ting, and genetic typing may be used to trace common source outbreaks.

By the use of these techniques, outbreaks from geographically distinct

regions have been linked.

Animals

Prevalence

Recently, NLVs have been found in cattle and pigs in the Netherlands.

The data suggested a very high prevalence of NLV in calf herds.

The animal viruses are genetically distinct from the NLVs found in

humans. However, the genetic distance is relatively small, suggesting

the animal and human NLVs could intermingle.

FoodShellfish is notorious as a source of food-borne viral infections because it

actively concentrates viruses from contaminated water. Infectious

viruses can be detected in these filter feeders for up to 6 weeks without

any loss in quality to the shellfish. Depuration, a practice that may reduce

bacterial contamination, it is not very effective in reducing viral load of

shellfish. In the early months of 2001, NLVs have been detected in at least

2 batches of imported shellfish in the Netherlands. In addition to shellfish

many other food items (including desserts and vegetables) have been

associated with NLV outbreaks according to the literature. All food that

has been handled manually and not sufficiently heated subsequently may

be a possible source of infection.

Conclusion

Strict implementation of hygienic rules is probably the most important

preventive measure. Food handlers with gastroenteritis should immedia-

tely be removed from the food chain. Given the highly infectious nature of

NLV it is envisioned that guidelines should be developed that include the

occurrence of gastroenteritis in people (e.g. children) in contact with

persons working in critical points in the food chain.

For prevention of food-borne transmission it is essential that food items

are not grown or washed in water contaminated with faeces. However,

the globalization of the food market has hampered the implementation of

control measures to assure safe food. It is not clear whether routine

monitoring of food specimens for viral contamination will be feasible.

The currently used criteria for measuring water and food quality do not

correlate with the presence of pathogenic viruses.

group of strains. This indicates either that pigs and humans share com-

mon sources of infection or that pigs area vehicle for M. avium infections

in humans. A survey in 1998 at two farms with incidences of caseous

lesions of 40-50% of the slaughtered pigs revealed compost as a risk-

factor; compost is used in the Netherlands as a supplement for piglets

with diarrhoea. The feeding of compost has been forbidden by the

Production Board since July 2000. Such extremely high incidences are not

seen anymore, but the monitoring of caseous lesions at three big

slaughterhouses reveals a slight increase in the monthly percentage

affected pigs. To identify other risk factors farms, farms with a high

incidence have to be investigated.

2.5 Norwalk-like Caliciviruses

IntroductionThe human caliciviruses are assigned to two groups, the genera Norwalk-

like (NLV) and Sapporo-like (SLV). The NLV are also known as ‘small-

round-structured-viruses’ (SRSV) and the SLV as ‘typical caliciviruses’.

The two virus groups differ epidemiologically: the NLVs cause illness in

people of all age groups, whereas the SLV predominantly cause illness in

children.

Some groups of animal caliciviruses have a broad host range, and it has

been postulated that NLVs can occasionally be transmitted between

humans and animals. At present, however, there is no scientific proof.

Food-borne transmission of caliciviruses is well known for viruses in the

NLV genus. Within this genus a great diversity of virus types exists.

NLVs are transmitted by direct or indirect contact with contaminated

water, food or environment. NLVs can survive outside the host, are

resistant to common disinfectants and extreme pH fluctuations, and are

highly infectious.

Humans

Prevalence

NLV infections are among the most important causes of gastroenteritis in

adults and often occur as outbreaks, which may be food-borne.

Food-borne NLV outbreaks are often caused by an infected food handler.

Outbreaks of NLV gastroenteritis (not only food-borne) are common in

institutions such as nursing homes, hospitals, restaurants, etc.

In the Netherlands approximately 80% of the outbreaks of gastroenteritis

that are reported to municipal health services are caused by NLVs.

Food-borne outbreaks are also reported through a network of food

inspection services, and the role of NLV as a cause of these outbreaks is

currently under investigation.

Results of surveillance

Preliminary results from studies in the Netherlands suggest that NLVs

may also cause a significant number of the food-borne outbreaks. Based

on studies from the UK and the US, it has been estimated that up to 30% of

the food-borne infections may be caused by NLVs.


2.6 Trichinella

IntroductionIn Europe, four Trichinella species have been found in wildlife: T. spiralis,

T. britovi, T. nativa and T. pseudospiralis. Of these, T. spiralis is the most

important because it is able to maintain itself in the domestic cycles of

pigs and horses.

Trichinellosis has not occurred in the Dutch pig population since 1979.

In 1999, 19,554,094 pigs were examined at slaughterhouses by the artificial

digestion method. In March 1999, one suspected Trichinella larva was

found. This larva was confirmed morphologically as Trichinella spp. by the

National Reference Laboratory for Trichinella (RIVM). Identification of the

larva to species level was negative. In addition, the positive pooled sam-

ple was further investigated, but no positive diaphragms were detected.

The pooled sample of 100 pigs was traced to three pig farms. These farms

were inspected for hygiene standards and rodent control. All three farms

kept the pigs indoors under good management. No additional measure-

ments were taken besides the mandatory Trichinella testing using

artificial digestion of the slaughter animals.

Humans

Prevalence

In the Netherlands, no autochthonous cases of trichinellosis had been

recorded for decades until 1999, when seven cases of human trichinello-

sis were diagnosed, based on positive serology. Of these cases, six were

imported infections, three of which could be traced back to an outbreak

with infected pork in Montenegro (Yugoslavia). One case was autochtho-

nous and possibly a reactivation of an old infection. In 2000, two human

cases were reported; both were imported infections.

Animals

Prevalence/investigation

The prevalence and identification of Trichinella infections in wildlife in

the Netherlands were studied from 1998 to 2000 at the RIVM (Table 2.6.1).

Wild boars were tested serologically using ES-ELISA and by artificial

digestion of muscles samples of diaphragm. A total of 577 blood samples

of wild boars (Sus scrofa) were tested. Of these samples, 35 were positive

for Trichinella antibodies. A total of 11 muscle samples were tested by

artificial digestion in 1999. Two muscle samples contained Trichinella

larvae (0.2 and 0.3 larva per gram). The Trichinella Reference Centre (TRC)

in Rome identified the larvae as T. spiralis. In 2000, 47 muscle samples

were tested by artificial digestion. Of these, only one sample was positive

(0.06 larva per gram). It was not possible to identify this larva to species

level.

In addition, 541 foxes (Vulpes vulpes) were tested by artificial digestion of

foreleg muscles. An overall prevalence of 5.5% was found. Only some of

the larvae could be identified. These were all T. britovi. Until now no

T. spiralis has been identified in foxes in the Netherlands.

In 2000, from the wild boars shot at the Veluwe, 882 animals were

investigated. All these animals were investigated by the RVV as part of the

regular inspection of meat in slaughterhouses. None of them were found

positive for Trichinella spp.

Conclusion

These investigations confirm the existence of two different Trichinella

spp. in wildlife in the Netherlands. The existence of sylvatic trichinellosis

has no consequences for industrialized pig farming. However, the presen-

ce of T. spiralis in the sylvatic cycle can indicate a potential risk of new

foci of domestic trichinellosis arising, mainly in organic pig farming.


Table 2.6.1 Prevalence of trichinellosis in foxes and wild boars during 1996–2000

Animals Tested Test Number of positives/ % Trichinella

species material total number tested species

Wild boar Blood ELISA 35/577 6.1

Wild boar Diaphragm Artificial 3/58 5.1 T. spiralis

muscle digestion

Red fox Foreleg muscle Artificial 30/541 5.5 T. britovi

digestion

Wild boar1 Diaphragm Artificial 0/882 0

muscle digestion

1 Investigated by RVV

General-practice-based and community-based epidemiological studies

In the general practice study from May 1996 to May 1999 (Chapter 1),

Yersinia was included in the case-control study component. Yersinia was

found in 6 patients (0.7%) and in an equal number of controls (1.1%), of

which most were Y. enterocolitica (all apathogenic types) (Table 2.7.1).

The age of the Yersinia-positive patients varied from 5 to 74 years (median

29.5 years) and from the positive controls from 42 to 72 years (median 53.5

years).

In the Sensor population-based study Yersinia was included in the faecal

analyses of the case control component (Chapter 1 and Frame 1). Yersinia

was found in 3 patients (0.4%) and in 5 controls (0.8%), of which most

were Y. enterocolitica (all apathogenic types, see Table 2.7.1). The age of

the patients with Yersinia in the study was 18, 35 and 77 years and from

the controls varied between 0 and 34 years (median 28 years).

AnimalsIn the Netherlands, pigs are the only known source of the pathogenic

serotypes O:3 and O:9 of Y . enterocolitica. A survey in 1998 showed that

22% of the porcine tonsils and 6% of porcine faeces were positive for

Y. enterocolitica, the majority being of serotype O:3 (Table 2.7.2).

2.7 Yersinia enterocolitica

IntroductionYersiniosis resulting from infections with Yersinia enterocolitica is a relati-

vely rare cause of diarrhoea and abdominal pain or cramps in Western

countries, but may lead to serious disease with rectal bleeding and ileum

perforation. It is also associated with serious sequela, such as acute and

chronic arthritis. More than 50 serotypes and 6 biotypes are recognized

as occurring in farm animals, rodents and pet animals. A minority of these

are pathogenic to humans: predominantly serotypes O:5, 27, O:8 (mainly in

the US, rarely in the Netherlands), O:3 and O:9, and biotypes 1B, 2, 3, 4 and

5, all often isolated from pig tongues and tonsils. From the end of the

eighties control has been directed at pig slaughtering procedures and

preventing contamination of carcasses by tonsils and tongues.

Humans

Results of laboratory surveillance

The number of Y. enterocolitica isolates reported by 15 regional public

health laboratories (see Chapter 1) halved within two years, from 207 in

1989 to about 100 per year in the period 1991-1996. As in neighbouring

countries, this was related to changes in pig slaughtering procedures and

preventing contamination of carcasses by tonsils and tongues. Serotype

O:3 was observed most frequently, with an annual proportion varying

between 51% and 64% of all isolates. Other relatively important types

were O:9 and in recent years also O:6 types. More than 95% of the isolates

originated from faeces. Most Yersinia isolates were found in young

children and, to a lesser extent, young adults. The total number of

laboratory-confirmed cases for the whole of the Netherlands is estimated

at 180 per year.


Table 2.7.1 Yersinia in patients and controls in the NIVEL GP-based study and Sensor population-based study

Agent NIVEL GP-based study (1996-1999) Sensor population study (1999)

Patients Controls Patients Controls

N=857 % N=574 % N=700 % N=665 %

Yersinia spp. 6 0.7 6 1.1 3 0.4* 5 0.8

Y. enterocolitica 5 0.6 5 0.9 3 0.4 3 0.5

Y. frederiksenii 1 0.1 1 0.2 0 1 0.2

Y. bercovieri 0 1 0.2

* After standardization for age, gender and cohort, this percentage was estimated at about 2%

38

Table 2.7.2 Isolation of Yersinia spp. from pigs

Material Yersinia spp. Positive/ % Frequency of serotype (biotype)

tested

O:3 O:9 Other

faeces Y. enterocolitica 9/149 6 5 1 3

tonsils 34/153 22 28 5 1

pork Y. enterocolitica 14/412 3 4 (type 4) 10 (type 1A)

Y. frederiksenii 3/412 1

FoodYersinia spp. can be isolated from a great variety of foods. However, these

isolates usually belong to non-pathogenic sero- and biotypes. Potentially

pathogenic Y. enterocolitica strains were isolated from 10% of pork

samples (Table 2.7.2). For the prevention of food-borne yersiniosis it is

essential to exclude contamination of pig carcasses with faeces or from

the tonsils.

Conclusion

Based on the available data, the occurrence of Yersinia at the end of the

eighties and in the early nineties decreased and subsequently stabilized

at a low level. Compared with other microorganisms, Yersinia now seems

to be of limited importance in the development of gastroenteritis in

humans.

2.8 Listeria monocytogenes

IntroductionIn the genus Listeria, human illness is predominantly caused by L. mono-

cytogenes, especially the serotypes 1/2a, 1/2b and 4b. The bacterium is

found in humans and a variety of animal species, but is also widespread in

the environment. Most human infection is caused by food (of animal

origin).

HumansSince 1987, 15 regional public health laboratories have been reporting

first isolates of Listeria spp. each week, with an estimated coverage of the

Dutch population of 62% (see section 1.1). Between 1991 and 2000 the

incidence of laboratory-confirmed infections with Listeria spp. was


consistently low, on average one case per 0.5 million inhabitants per year,

or 31 cases per year for the whole of the Netherlands. Cases were found

more often among young children, at one case per 300,000 children

0-4 years old, and older people, at one case per 150,000 inhabitants

60 years and older. Twice as many cases were found in women

15-44 years of age than in men of comparable age, but the numbers con-

cerned are low. Between 1995 and 1998 findings were about 70% higher

than in the following and preceding years. Serotyping was reported in

16% of the isolates and showed no evidence of a shift in serotype

distribution between 1991 and 2000, consisting predominantly of 4b (51%)

followed by 1/2a (23%), 1/2c (16%) and 1/2b (10%). Of these positive cases,

65-70% were isolated from blood and about 10% from liquor, which might

be considered as proxies for cases with septicemia and meningitis

respectively. The national reference laboratory for Listeria spp. at RIVM

typed 89 L. monocytogenes isolates from blood and liquor from patients all

over the country (35, 28 and 26 cases in 1998, 1999 and 2000 respectively).

In this selection, serotype 4b again dominates (40%), followed by 1/2a

(30%), 1/2b (24%) and 6% other types such as 1/2c, 3a, 3b and 4e.

AnimalsThe bacterium is found in almost all vertebrates, especially ruminants.

The clinical infection of animals differs from asymptomatic to acute

symptoms (including abortion). The bacterium is secreted into the animal

faeces. In slaughterhouses these faeces can cause cross-infection of

the carcass. This environmental contagion in the food industry (including

water supply, cold store) can cause originally untainted products to

become infected. Good hygiene management is indispensable in the

slaughterhouses and the food industry.

cases occurred annually until 1995. The last infected herd was culled in

1996. In August 1999 the Netherlands was declared officially free of

bovine brucellosis by the European Commission.

Brucella melitensis has never been reported in animals in the

Netherlands.

The Netherlands is also free of B. suis. Only two cases were reported in the

late sixties and two in 1973. Three of these cases were imported infected

pigs and one was caused by feeding infected offal from imported hares.

Maintaining the official free status

Brucella bovis

Before 1999 the surveillance system and control of outbreaks of brucello-

sis in cattle were carried out autonomously by the Animal Health Service

under regulations of the Production Board of the industry. The surveillan-

ce system was primarily based on monthly testing of all dairy herds with

the milk ring test and a yearly serological testing of all animals older than

12 months in non-dairy herds.

On October 1st 1999 the control of outbreaks was placed directly under

the responsibility of the Ministry of Agriculture Nature Management and

Fisheries by a change of regulations under the Animal Health and

Welfare Act. From that date the RVV has been responsible for controlling

outbreaks of brucellosis.

Monitoring is still undertaken by the Animal Health Service, under respon-

sibility of the ministry. According to Directive 98/46 all animals older than

24 months in 20% of the present cattle herds are tested only once a year.

Dairy farms are tested by analysing bulk milk tank samples with an ELISA

test. Positive samples are re-tested with the milk ring test. Individual

blood samples are taken of herds on non-dairy farms and tested by a

microagglutination test. Positive samples are re-tested with an ELISA test.

Blood of cows that have aborted are first tested with a microagglutination

test. Positive samples are re-tested by the complement fixation test.

No cases of bovine brucellosis were diagnosed. However, false positive

tests led to 54 farms being unnecessary for about 2 months quarantined in

2000. These false positive tests were mainly due to the testing of aborted

animals.

Brucella melitensis

To maintain the official free status an annual screening programme under

Decision 94/953 has been set up to prove with a confidence of 95% that

less than 0.2% of sheep and goat herds are infected with B. melitensis.

All samples gathered in this screening programme since 1994 have

proved to be negative.

Brucella suis

The free status is maintained by the notification system. According to

Directive 90/429 boars are tested before entering and when leaving the

semen centre. Breeding animals exported to non-member states are also

tested. All animals tested were negative for B. suis.

FoodThe Inspectorate for Health Protection, Commodities and Veterinary

Public Health monitors the occurrence of Listeria monocytogenes in

foods in retail outlets. According to the Dutch Commodity Act, tests must

be carried out on food products prepared for direct consumption.

The bacterium should be absent in 0.01 g of the product (<100 bacteria per

gram of product). The results of the monitoring carried out in 1999 are

reported in Table 2.8.1.


Table 2.8.1 Percentage distribution of the number of foods in

retail outlets containing >100 cfu/g Listeria

monocytogenes in 1999

Sample type Number of positives

/Number of samples

> 100/g (%)

Heat-treated products of pork,

beef, chicken and turkey 11/1373 0.8

Non-heat-treated products of pork

beef, chicken and turkey 8/999 0.8

Smoked, salted, non-heat-treated

or slightly heat-treated fish products 3/558 0.5

Sprouts or sliced vegetables 0/573 0.0

Cheese and cheese products 1/132 0.7

2.9 Brucella

IntroductionCases of brucellosis in humans and animals are notifiable in the

Netherlands. According to EU legislation the Netherlands was declared

officially free of bovine brucellosis caused by B. abortus in 1999 and of

brucellosis caused by B. melitensis in sheep and goats in 1992.

The Netherlands is also free of B. suis.

HumansIn the Netherlands brucellosis in humans is notifiable for laboratory

diagnosis. From 1 to 4 cases of people infected abroad are reported each

year. Three cases were reported in 2000.

Animals

History

Bovine brucellosis used to be endemic in the Netherlands. Since the

Dutch national Brucella abortus control programme for cattle started in

1959 its prevalence has dropped from about 30% to 1% in 1964. Sporadic

Table 3.1.1 Animals Investigated for rabies in 1999 and 2000

Animal species Number of positives/ total number tested

1999 % 2000 %

Bats 6/57 11 3/89 3

Foxes 0/6 0 0/8 0

Dogs 0/3 0 0/2 0

Cats 0/5 0 0/9 0

Squirrels 0/10 0 0/0 0

Mice 0/1 0 0/1 0

Hyenas 0/1 0 0/0 0

Donkeys 0/1 0 0/0 0

Rats 0/0 0 0/1 0

3.1 Rabies virus

IntroductionThe rabies virus belongs to the Rhabdoviridae family (genus Lyssavirus).

In Europe two types of this virus are important: the classical virus (street

virus) and the European Bat Lyssa (EBL) virus, which can be subdivided

into various subtypes. In Northern Europe the EBL virus subtype 1a

(EBL-1a) is endemic in free-living insectivorous bats. The virus is almost

exclusively present in one bat species, Eptesicus serotinus.

The street virus is spread mainly by foxes. Nearly all the human rabies

cases worldwide are caused by this street virus. The EBL -virus, however,

can infect humans too; in Europe some fatal cases caused by this virus

have been reported.

HumansPeople that have been in contact with animals diagnosed as rabid are

given post-exposure treatments vaccinations and vaccinations combined

with application of anti-rabies immunglobulin. This is also applied when a

suspected contact animal cannot be investigated.

The treatment can be carried out by general practitioners, who can obtain

information about (the necessity) of the treatment at the National Poisons

Control Centre (NVIC) of the RIVM.

No human cases were reported in 1999 and 2000. In 1999 information

about 34 cases of post-exposure treatment was provided by the NVIC.

The majority of these cases (22) were related to contacts with bats.

The other cases were related to contacts with cats (3), squirrels (3),

a ferret, a dog, a mole, a mouse, a rat and a fox. In 2000, this information

was given 37 times in the Netherlands. In 32 of these cases, the vaccina-

tion was indicated after exposition from persons to a possibly rabid

animal.

Animals

Prevalence

Until 1988 the street virus was occasionally diagnosed in foxes in the

border regions of the Netherlands as a result of contacts with foxes in the

neighbouring areas in Germany and Belgium. After long-term vaccination

campaigns in these areas and in the southern part of the Netherlands

during 1988-1992, the street virus has not been diagnosed in foxes in the

Netherlands.

Results of investigations

In the Netherlands wild animals, farm animals and pets displaying abnor-

mal behaviour (inexplicable aggression) are considered to be suspected

cases of rabies. Investigation of these animals takes place at ID-Lelystad.

During 1999 a total of 84 animals were investigated for rabies (Table 3.1.1).

Six bats (all belonging to the species Eptesicus serotinus) were found

rabies positive. One bat originated from Finland. Eight investigated

squirrels were imported from China. The hyena belonged to Amsterdam

Zoo. In 2000 a total of 110 animals were investigated for rabies (8 foxes,

2 dogs, 9 cats, 1 rat, 1 mouse and 89 bats). Three bats were diagnosed

rabid.


Chapter 3

Direct and Environment-Mediated Zoonoses

Characterization of a newly detected lyssavirus and study of

transmission in frugivorous zoo bats

In July 1997 a lyssavirus was isolated from a colony of Egyptian flying

foxes (Rousettus aegyptiacus) originating from Rotterdam Zoo, sparking

discussions about the public health hazard of the virus and its reservoir

hosts. Rotterdam Zoo had kept a large colony of Rousettus aegyptiacus

bats in a large artificial cave exhibit for more than 6.5 years. Since the

establishment of the colony, rabies had never been detected and all dead

bats had been submitted for rabies control. It is a mystery how the virus

was introduced into the colony because no new bats were introduced

after establishment in the cave, and contact with free-living animals was

not possible.

Investigations and results

The lyssavirus infection was first detected in two bats that had died short-

ly after export to the Ødense zoo in Denmark. Sequencing of two major

parts of the genome of this newly isolated virus revealed that the virus

was an EBL-1a. To elucidate the characteristics of this isolated lyssavirus,

experimental intracranial infections with the Danish lyssavirus isolate

were performed in frugivorous bats and mice, and current lyssavirus

detection assays were evaluated. The virus induced neurological signs in

both species and its pathogenicity in bats suggested that like many other

mammals, Rousettus aegyptiacus bats could be victims of EBL-1a infecti-

ons, besides reservoir hosts. After the experimental infection in 16 bats,

B. henselae infections, development of specific and sensitive serology is

required. Based on the number of positive tests, Bergmans et al.

estimated in 1997 that about 2000 new cases arise each year. This would

account for an incidence of about 12.5 per 100,000 inhabitants.

Usually the disease is self-limiting, and causes a benign but chronic

inflammation of the lymph nodes afferent from the scratch or bite.

Very often young children are the victims of the affliction, which usually

lasts for several months and can cause considerable pain. Antibiotic

treatment is ineffective and in many instances the lymph node needs to be

drained or even surgically removed. In some cases, particularly in

patients with an impaired immune system, systemic infections occur

which may afflict several organs, particularly the liver and spleen.

However, systemic infections can be treated successfully with antibiotics.

Animals


In the Netherlands there is no surveillance system to detect Bartonella

infections in animals. There has been only one study, performed by

Bergmans et al. (1996), in which blood from pet cats in animal sanctuaries

was sampled and analysed by culture, PCR and serology.

In 1997, Bergmans et al. showed that 22% of the asymptomatic pet cats

investigated were bacteremic for B. henselae. Over 50% of the cats were

serologically positive. If there are about 2 million pet cats in the

Netherlands and approximately a quarter of them are infected with

B. henselae they represent a significant reservoir of this pathogen.

Antibiotic treatment can eliminate the bacterium from the cats systems

and after treatment cats may be protected against re-infection. However,

large-scale vaccination of pet cats would probably be the best way to

reduce the size of the reservoir for B. henselae and significantly reduce

the number of cases of cat scratch disease.

Conclusion

Due to the high number of infected cats and the relatively high incidence

and young age of human patients, cat scratch disease is an important

zoonotic disease.

3.3 Borrelia, Ehrlichia and Babesia

Introduction

Of the many different tick species, the most prevalent in the Netherlands

is Ixodes ricinus. This tick will bite many different hosts, ranging from

reptiles to birds and mammals, including humans, and can transmit a

variety of pathogens from one host to another. In Europe I. ricinus is the

predominant carrier and transmitter of Borrelia burgdorferi, the causative

agent of Lyme disease. In the last decade, several researchers have

shown that 10–35% of the Dutch I .ricinus ticks are infected with

B. burgdorferi sensu lato. This infection rate shows regional differences.

Although it is clear that birds, small rodents and roe deer are competent

reservoir hosts for B. burgdorferi sensu lato. It is unknown what the rate

of infection of these animals in the Netherlands is.

all current assays – mouse inoculation test (MIT), immunofluorescence

test (IF), RT-PCR and immunoperoxidase staining (IP) – readily detected

the lyssavirus infections in frugivorous bats.

To study the transmission of EBL-1a in a bat population, 16 bats in a group of

32 frugivorous bats were intramuscularly inoculated with the virulent

Ødense EBL-1a isolate. Inoculated bats were randomly mixed with mock

infected sentinels. Subsequently, all bats of the group were regularly

sampled and monitored for two months using lyssavirus antibody-, antigen-

and RNA detection assays. Primary EBL-1a infection was detected in 25%

of the intramuscularly inoculated bats. Actual transmission of EBL-1a could

be detected in no more than 13% of sentinel bats. It may be concluded that

intramuscular EBL-1a infection was not very effective, and that EBL-1a was

not rapidly transmitted within a colony of frugivorous bats.

Conclusion

From these two experiments it was concluded that the newly isolated

lyssavirus is a virulent and pathogenic strain. Nevertheless, the virus did

not spread rapidly after experimental infection in a bat population.

Because of the endemicity of rabies in free-living bats in Northern Europe

it is recommended to avoid inhaling excreta from bats and not to touch

bats without protection. The result of these two experiments indicates

that the same preventive measurements should be taken regarding zoo

bats. Extra precautions such as pre-exposure treatments only seem to be

indicated for people who are in regular contact with bats, like research

workers and zoo keepers.

3.2 Bartonella henselae

IntroductionThe causative agent of cat scratch disease is Bartonella henselae.

The cat is the only known reservoir. Although infected cats are usually

bacteremic, with high numbers of live bacteria in their blood, they rarely

display clinical signs. Transmission between cats is almost certainly

mediated by cat fleas.

The exact mechanism of infection is unknown, but it is clear that

B. henselae is transmitted from infected cats to humans by traumatic

contact such as scratching or biting. There is no evidence that

transmission between humans does occur.

Humans


Due to the fact that this disease is not notifiable, no surveillance exists.

The true incidence of cat scratch disease in the Netherlands is unknown.

Diagnosis of cat scratch disease is difficult due to its broad clinical

spectrum and the unreliable serology. Culture of B. henselae from

affected lymph nodes or blood from cat scratch disease patients is

virtually impossible. However, PCR analysis of lymph nodes aspirates or

biopsies from afflicted lymph nodes or organs is highly sensitive and

specific, but it does require invasive methodology. In order to be able to

determine the prevalence and incidence and to improve the diagnosis of


Another tick-transmitted human pathogen is the agent causing human

granulocytic ehrlichiosis (HGE). This organism is closely related, if not

identical, to Ehrlichia phagocytophila and E. equi. These bacteria occasio-

nally cause tick-borne fever in ruminants and horses, respectively.

Finally, protozoan parasites belonging to the genus Babesia are the cause

of some sporadic cases of clinically manifest babesiosis in cattle.

Humans

Prevalence

Using postal questionnaires, de Mik et al. carried out a survey in 1994 of

nearly 80% of all Dutch general practitioners (GPS). This revealed that in

1994 the GPS saw 6,500 patients with clinically manifest cases of Lyme

disease, an incidence of 43 per 100,000 inhabitants. Furthermore, these

GPS also reported 33,000 patients with sustained tick bites, indicating that

the real number of humans with tick bites is huge. Repetition of the type of

survey de Mik et al. performed at a five-year interval may be helpful to

assess any trends in the epidemiology.

In 1999, the first and so far only case of endogenous human granulocytic

ehrlichiosis in the Netherlands was reported. However, the high degree of

ehrlichia infections in Dutch ticks would suggest that more human cases

of ehrlichiosis do occur. Therefore, increased awareness among

clinicians and improvement of the diagnostic tools to detect ehrlichia is

required. In this respect, a prospective study of blood samples from

patients that suffer from febrile diseases after a tick bite may be of value

to determine the true incidence of human granulocytic ehrlichiosis in the

Netherlands.

Fewer than 500 cases of babesiosis in man have been reported world-

wide, making it a relatively rare zoonosis. There have not been any

documented cases of endogenous tick-borne encephalitis or babesiosis

in Dutch patients.

Surveillance and results

Due to the fact that these diseases are not notifiable in the Netherlands, no

surveillance exists.

In 1999, 75 blood or serum samples from patients displaying clinical

features that might be indicative for ehrlichiosis were subjected to serolo-

gical and PCR analysis. None of the samples analysed contained any

detectable Ehrlichia DNA. Serology of these patients was inconclusive;

some samples were positive in the direct immune fluorescence assay and

others reacted to an immunoblot using a recombinant Ehrlichia antigen.

No conclusions on the prevalence of Ehrlichia infections could be drawn.

Animals


There is no surveillance system to detect any tick-borne infection in wild

and domestic animals in the Netherlands. However, several researchers

have determined the prevalence of infections with Borrelia burgdorferi in

Dutch ticks. In most surveys, ticks were collected by flagging from the

vegetation and will be investigated by culture, dark field analysis or PCR.

In 1999, Hovius et al. showed that all the 85 Dutch pet dogs included in

their study became infected with B. burgdorferi over a period of five

years. In addition, 24 of these dogs (28.2%) developed Lyme disease.

In 1999, Schouls at al. reported that 13% of the ticks collected from roe

deer shot in the Flevopolder carried B. burgdorferi. In addition, 45% of the

same ticks carried Ehrlichia species and more than half of these species

belonged to the E. phagocytophila genogroup, which includes species

that are potentially pathogenic to man. In a similar survey, nearly 10% of

the ticks were also found to carry Ehrlichia species. Siebinga et al.

reported in 1999 several cases of ehrlichiosis in cattle from a dairy farm in

Friesland. Systematic studies on the presence of tick-borne encephalitis

virus and Babesia in ticks have not been performed.

3.4 Hantavirus

IntroductionHantaviruses are enveloped viruses of the family Bunyaviridae, with a

three-segmented negative-sense RNA genome. Hantavirus infections

were brought to the attention of western physicians by the impact of an

infection among US soldiers, who developed a severe haemorrhagic

illness, during the Korean war. At least five distinct serotypes have been

described: the prototype Hantaanvirus, which is mainly documented in

Asia; Seoul virus, with a worldwide distribution; Puumalavirus, which is

predominantly endemic throughout Europe; Dobrava virus, which is

recognized in eastern and central Europe; and Sin Nombre virus, which is

documented in the Americas. Each serotype is associated with a single

rodent host species or genus and is transmitted to man via aerosols of via

contaminated rodent excreta.

There are two main types of disease described in humans: Haemorrhagic

Fever with Renal Syndrome (HFRS) caused by Puumalavirus, Hantaan-

virus, Seoul virus and Dobrava virus, and Hantavirus Pulmonary

Syndrome (HPS) caused by Sin Nombre virus. A mild form of infection by

Puumalavirus is known as Nephropathia Epidemica (NE) and is one of the

most common rodent-borne infections in Europe. Serological confirmation

is needed when HFRS or HPS is diagnosed.

Humans

Prevalence and results

In 1986 and 1988, the first two non-laboratory associated cases of

hantavirus infections were described in the Netherlands. However,

retrospective studies of specimens from patients with acute interstitial

nephritis showed that cases of hantavirus in the Netherlands could be

traced back to 1974. Until 1995, 39 hospitalized cases of serologically

confirmed hantavirus nephropathy in humans, all caused by Puumala-

virus, have been documented in the Netherlands. The majority of these

cases occurred in the eastern and southern part of the country. Between

1996 and 1999, 750 serum samples of patients suspected of NE were

tested for the presence of Puumalavirus antibodies. In 41 patients (5.5%)

the clinical diagnosis of NE could be confirmed by positive hantavirus

specific serology. In the years from 1996 to 1999, hantavirus specific anti-


0

100

200

300

400

500

600

1998 1999

HSV HBV SIV STLV Ebola several

Also, a HBV virus from the blood of woolly monkeys was isolated by PCR.

On the basis of sequence analyses, this newly discovered HBV virus was

different from the human HBV and gibbon and chimpanzee hepatitis virus.

As a result of this, all HBV antigen positive animals were brought to

another facility where there was no contact with the public.

These preliminary surveillance data on primates housed in zoos and

biomedical research centres in the Netherlands have already resulted in

the development and implementation of uniform primate health guidelines

and guidelines for primate animal-carers.

bodies could be detected in 28, 4, 2 and 7 serum samples from NE

patients. The percentage of hantavirus-positive patients varies between

1.4% and 5.5% per year. This is in agreement with previous findings in the

Netherlands during the last decade.

Animals

Results

In Western Europe, hantavirus antibodies and antigen have been

demonstrated in non-rodent species, for example in bats and cats.

Hantavirus-specific serological tests on domestic and feral animals for

the presence of hantavirus, performed in the Netherlands in 1995, only

demonstrated the presence of this virus in the red bank vole

(Clethrionomys glareolus), the common shrew (Crocidura russula) and the

common vole (Microtus arvalis), which all demonstrated Puumalavirus

antigen in their lungs. Puumalavirus was only isolated on cell-culture from

the lungs from the red bank vole. All positive animals were trapped in

areas where human cases of NE had been identified.

3.5 Viruses in primates

IntroductionHumans and primates share susceptibility to many pathogens, due to their

close phylogenetic relationship. This close relationship between humans

and primates makes these animals invaluable models for studying human

infectious diseases, but it also makes them potentially dangerous to work

with. In collaboration with the Society of Zoo Veterinarians and W&V/KvW,

human and non-human primates were tested for the presence of viral

zoonotic antibodies, including herpes simplex virus (HSV), simian immuno-

deficiency virus (SIV), simian T-cell leukemia virus (STLV), hepatitis B virus

(HBV) and Ebola virus. These epidemiological studies are carried out to

investigate the presence of these viruses in primates in the Netherlands

and to develop new guidelines for animal caretakers and authorities.


A serological surveillance programme for the detection of these viruses

was started in 1996. The blood samples of the primates from the

respective zoos are taken during their yearly check-up for tuberculosis or

when other health problems with the animals occur.

The number of primates tested in 1998 and 1999 were 1,431 and 1,408,

respectively, and 1,912 and 1,963 serological analysis were performed

(Figure 3.5.1). No antibodies against SIV and Ebola virus were detected in

any of the monkeys. In 1998 and 1999 STLV antibodies were detected in

several macaque species (n = 9). HSV virus is used as a serological

marker for herpes B virus. If monkeys are HSV positive their antibodies

were re-tested by specific herpes B virus-blocking immunoassays or

virus neutralization tests for the presence of antibodies specific for

herpes B virus. In 1998, 24 (11%) t of 214 serum samples tested positive

for HSV-specific antibodies. In 1999, 21 (19%) of 111 samples were

HSV antibody positive. In none of the HSV seropositive animals could

clear evidence of herpes B virus specific antibodies be demonstrated.


Figure 3.5.1 Comparison between the primates screening in 1998

and 1999

3.6 Echinococcus

IntroductionEchinococcus granulosus is a tapeworm of dogs. The larval stage occurs

in domestic animals, such as cattle, sheep and horses. In man a similar

hydatid stage is diagnosed.

E. multilocularis is a serious parasitic zoonosis. The parasitic life cycle of

this tapeworm is mainly sylvatic. Eggs shed by the canid definite host,

mainly the red fox in Europe, develop to the larval or metacestode stage

after uptake by arvicolid rodents, which serve as intermediate hosts.

However, in accidental cases humans may get infected by ingesting the

eggs and this can lead to a very serious disease, alveolar echinococcosis.

Although the transmission routes to humans are not clearly understood

(the relative role of foxes, dogs and cats) and the susceptibility for

humans of this parasite is assumed to be limited, measures to prevent

human infection are of major importance.

Humans

Prevalence

In 1999 there were 31 imported human cases of E. granulosus in the

Netherlands.

Until now, there has been only one human case of alveolar echinococcosis

in the Netherlands. This person was born in Switzerland and caught the

infection in that country, which is one of the high endemic areas in Europe.

Table 3.7.1 Results of investigations for (embryonated)

Toxocara eggs in soil

Public parks (1993): 10–30% mainly T. canis

Sand boxes (1993): 30–70% mainly T. cati

Potting soil (1998): 15% T. canis + T. cati

Table 3.6.1 Results of the study of E. multilocularis

in foxes 1996-2000

Area Number of positives/

total number tested

Border 5/272

Veluwe 0/72

Coastal 0/109

Groningen 10/106

Animals

Prevalence

In the Netherlands only the minor pathogenic strain of E. granulosus in

cattle occurs, but there are no recent data available about the prevalence

in cattle.

The determination of the prevalence of E. multilocularis in definite hosts is

an important parameter for estimating the potential infection risk to

humans in endemic areas. In the Netherlands, E. multilocularis was never

found in foxes before 1996.

From 1998 to 2000, a study in Groningen, one of the areas where

E. multilocularis was detected in foxes, was carried out to determine the

prevalence in that area, with the aim of evaluating trends in time and the

potential risk to humans. The results of 106 tested foxes showed that the

prevalence in this area is 9.4 %. These data indicate that E. multilocularis

is present in the Netherlands and that in Groningen the prevalence in

foxes is of such concern that periodical monitoring is appropriate.

The presence of E. multilocularis in its larval stage in the muskrat was

also demonstrated in Groningen, although at a very low level. In

1998–1999, 1,200 musk rats were investigated and only one found positive

(0.1%). In the same period, 526 muskrats from the southern part of Limburg

were investigated and all were found to be negative for E. multilocularis.


In a study carried out between 1996 and 1998, E. multilocularis was

demonstrated in 5 of 272 foxes in two distinct areas close to the border

with Germany and Belgium. Two positive foxes were found in the northern

province of Groningen and 3 positive foxes were found in the southern

province of Limburg. Future studies carried out in 1998 and 1999 in the

Veluwe (central part of the country) and the coastal areas resulted in no

new cases of E. multilocularis. The results of the study of foxes between

1996 and 2000 are shown in Table 3.6.1.

3.7 Toxocara

IntroductionToxocarosis is the clinical disease in man caused by infection of zoonotic

roundworms of dogs and cats, Toxocara canis and Toxocara cati.

The main mode of transmission to humans is oral ingestion of Toxocara

eggs from the environment. Direct contact with animals is not considered

a potential risk. Children more frequently have clinical symptoms because

of their closer contact with contaminated soil in gardens and sand pits,

the lack of hygiene and of eating soil. Toxocara larval migration in the

body can cause various clinical syndromes. Toxocara infections lead to

the elevation of serum IgE concentration, the presence of allergen-speci-

fic IgE and eosinophilia. In one of the epidemiological studies there was a

correlation between Toxocara seroprevalence and the onset of asthma.

It was concluded that in asthmatic children, infection with Toxocara may

boost allergic manifestations.

Humans

Prevalence

A survey among 800 healthy individuals in the Netherlands (PIENTER

Project, RIVM: 1995) revealed that 10% of children up to 10 years of age

and 30% of adults had antibodies against Toxocara. This reflects an

infection with the larvae of this parasite. Toxocara larvae have the ability

to survive in the tissues for at least 10 years. An average seroprevalence

of 19% in humans corresponds to 3 million infected Dutch inhabitants.

Even if only 1% of this group showed clinical disease, this would amount

to 30,000 patients per year. The actual number of cases is therefore

probably much higher than expected by physicians, because toxocarosis

has been proven to be an unknown disease and there is no national

mandatory reporting. It can be concluded that Toxocara and toxocarosis

may constitute a significant health problem.

Environment

Prevalence

Several studies from all over the world have demonstrated high rates

(10-30%) of soil contamination with Toxocara eggs in parks, playgrounds,

sand boxes and other public places. In the Netherlands (Utrecht)

comparable studies were carried out and results collected (Table 3.7.1).

Non-published results showed that eggs can also survive composting.


influenza A virus strains. Positive samples were used for virus isolation in

embryonated chicken eggs. The isolated viruses were typed with a panel

of H- and N-specific antisera. Although the subtyping of most of these

viruses could be achieved in this way, four viruses could not be typed by

this method. It is tempting to speculate that these viruses exhibit a not

previously recognized HA subtype (H16?). This further stresses the

importance of the surveillance of influenza in birds.

Following the surveillance studies, classical reassortment and reverse

genetics experiments were started for the exchange of gene segments

between influenza A viruses. These experiments are continuing. This may

allow the identification of gene products to enable the replication of avian

influenza viruses in mammalian (human) hosts and vice versa. These

studies may lead to the identification of patterns and requirements for the

transmission of influenza virus subtypes between different species, which

will eventually lead to predictions about avian influenza virus subtypes

that pose a hazard to public or animal health.

This project and a number of its ongoing extensions are currently being

conducted in close collaboration with Dr G. Koch (ID-Lelystad: LNV

funded project 47297.00), who focuses on similar surveillance and

experimental infection studies in domestic fowl, using essentially the

same techniques.

Besides these studies, a candidate vaccine against avian influenza

A(H5N1) virus was made and evaluated in a chicken model. In addition,

studies on the prevention and treatment of avian influenza (H5N2) influen-

za in a mouse model were carried out. Finally, a monkey model for avian

influenza A(H5N1) virus was established, which may serve for future

pathogenesis and vaccine/therapy studies.

2 Influenza B virus in seals Influenza B virus is a human pathogen whose origin and possible reser-

voir in nature are not known. An influenza B virus was isolated from a

naturally infected harbour seal (Phoca vitulina) and was found to be

infectious to seal kidney cells in vitro. Sequence analyses and serology

indicated that influenza B/Seal/Netherlands/99 is closely related to virus

strains that circulated in humans 4 to 5 years earlier. Retrospective analy-

ses of sera collected from 971 seals showed a prevalence of antibodies to

influenza B virus in 2% of the animals after 1995 and in none before 1995.

This animal reservoir, harbouring influenza B viruses that have circulated

in the past, may pose a direct threat to humans.

3 Swine influenza A virusesTo explore the occurrence of antigenic drift in swine influenza A(H3N2)

virus, we examined virus strains from outbreaks of respiratory disease

among finishing pigs in the Netherlands in 1996 and 1997 and from earlier

outbreaks. In contrast to swine H3N2 strains from the 1980s, the recent

isolates did not show significant cross-reactivity with human influenza

A(H3N2) viruses from 1972-1975 in haemagglutination inhibition tests.

These new strains form a separate branch in the phylogenetic tree of the

HA1 parts of HA. We conclude that recently there has been considerable

antigenic drift within the swine H3N2 viruses in the Netherlands and

Belgium (13).

AnimalsThe actual prevalence of patent Toxocara infections in the Dutch dog and

cat populations was investigated in several field surveys during the

period 1994–1997. The results are presented in Table 3.7.2. The percenta-

ge of positive faeces samples was in general significantly higher in young

animals than in older animals. Even a relatively low percentage of 5-10%

infected animals should not be neglected, because even that figure repre-

sents more than 200,000 Dutch dogs and cats, which daily shed billions of

Toxocara eggs into the environment.


Table 3.7.2 Results of investigations for the actual prevalence

of Toxocara infections in dogs and cats

(faeces samples positive for Toxocara eggs)

Category Number of positives / % T. canis % T. cati

total number tested

Privately-owned dogs 10/272 4 -

Privately-owned cats 11/236 - 5

Breeding dogs 46/445 10 -

Breeding cats 4/337 - 1

Stray cats 12/56 - 21

Conclusion

Human toxocarosis is considered to be one of the most prevalent parasi-

tic zoonoses from dogs and cats in the Netherlands. The number of dogs

and cats infected with the parasite indicates that preventive measures

are important and consist of preventing contamination of the environment

with Toxocara eggs. Education of the public is required to increase

awareness about this zoonosis.

3.8 Influenza

1 Avian influenza A viruses After the first identification of a fatal influenza A(H5N1) virus infection in

humans, which prompted us to issue a ‘pandemic warning’, the importan-

ce of surveillance of avian influenza viruses in wild and domestic birds

became even more evident than before. This prompted us to initiate a

feasibility study for a project to collect cloaca samples from several avian

species in the Netherlands. The initial 85 samples from the Krimpener-

waard yielded 10 paramyxoviruses and six influenza A viruses of the H6N1

subtype, as shown by serological and molecular analyses. These H6N1

viruses proved to have a haemagglutinin cleavage site indicative for a

non-pathogenic phenotype. Encouraged by these preliminary data,

we intensified the sampling of birds and analysis of avian influenza

viruses. To this end, organizations which for professional or other reasons

regularly have contacts with wild and migratory birds were approached

and asked to collect cloaca swabs or faecal samples. About 6,000 samp-

les were collected from more than 10 bird species. These samples were

first tested for the presence or absence of influenza viruses with a newly

developed PCR method, which allows the detection of all currently known

To explore the occurrence of antigenic drift in swine influenza A(H1N1)

viruses, we examined 26 virus isolates from outbreaks of respiratory

disease among finishing pigs in the Netherlands in the 1995/1996 season

and reference strains from earlier outbreaks using antigenic and mole-

cular methods. In contrast to swine H3N2 influenza viruses isolated from

the late 1980s to 1996, we did, however, detect a marked antigenic and

genetic heterogeneity in haemagglutination inhibition tests and nucleo-

tide sequence analyses among the 26 swine H1N1 influenza virus strains

isolated during our 1995/1996 surveillance. Interestingly, the observed

antigenic and molecular variants were not randomly distributed over the

farms. This finding indicates independent introductions of different swine

H1N1 influenza virus variants on the various farms in the study, and points

to the existence of a marked difference between the epidemiologies of

human and swine influenza viruses. The observed heterogeneity indicates

the necessity of regular monitoring of the antigenic reactivity of influenza

viruses from swine.

It is generally believed that pigs can serve as an intermediate host for the

transmission of avian influenza viruses to humans or as mixing vessels for

the generation of avian-human reassortant viruses. Here we describe the

antigenic and genetic characterization of two influenza A(H1N1) viruses,

which were isolated in the Netherlands from two patients who suffered

from pneumonia. Both viruses proved to be antigenically and genetically

similar to avian-like swine influenza A(H1N1) viruses which currently

circulate in European pigs. It is concluded that European swine H1N1

viruses can infect humans directly causing serious disease without the

need for any reassortment event.

References can be requested by the E-mail of the author.


The animal strains were isolated in the zoonosis monitoring programme of

RIVM. Samples were taken at farms; one isolate per serotype of

Salmonella for each farm. In the animal isolates S. Typhimurium was the

predominant serotype. DT104 was only isolated in small numbers: once in

1998 and 16 times in 1999.

The human Campylobacter strains were all clinical isolates. The animal

strains were isolated in the zoonosis monitoring programme of the RIVM

and from faecal samples taken at slaughter from healthy animals.

Microbiological methodsE. coli and E. faecium were isolated on MacConkey agar and Slanetz and

Bartley agar, respectively, by inoculating the plates with 50 µl of serial

dilutions of the sample in saline with a spiral plater. A colony with typical

morphology was pure cultured and typed biochemically. Campylobacter

was cultured on CCDA agar with 32 µg/ml cefoperazone and 10 µg/ml

amphotericine B to inhibit growth of Gram-negative bacteria and fungi.

Susceptibility was tested quantitatively with the micro broth dilution test

according to NCCLS guideline (M7-A3). For this test Sensititre trays were

used with custom-made panels of antibiotics (see Tables 1 to 5).

These microtitre trays were manufactured by Accumed Ltd in the UK.

ATCC strains were used to control the quality of the results. Strains with

MICs higher than the breakpoints were considered resistant. Calculations

of the proportions of resistance were based on NCCLS breakpoints and

breakpoints used in the Danish Monitoring Programme (DANMAP).

Results

E. faecium (Table 4.1.1)

• In 1999, after the use of tylosin as an antimicrobial growth promoter

(AMGP) was banned, proportions of resistance to macrolides were

lower.

• Vancomycin resistance was highest in strains from veal calves (42%).

However, the majority of these strains were isolated before the ban

on avoparcine. In 1998 only 25 E. faecium strains were isolated from

veal calves. Only one was vancomycin resistant (4%) indicating a

reduction as a result of the ban.

• Acquired resistance to ciprofloxacin is described in human clinical

isolates. The MICs of the E. faecium strains tested vary from 0.25 to

16 µg/ml. The distribution is monomodal and it seems that this is the

normal MIC level of ciprofloxacin for E. faecium. Little evidence for

acquired fluoroquinolone resistance exists.

• Nitrofurantoin MICs are all very high. DANMAP used 64 µg/ml as

breakpoint and calculated proportions of resistance. From the study

of the MIC distributions of nitrofurantoin no resistant sub-populations

could be detected and it seems as though the normal susceptibility of

E. faecium is displayed and all strains are intrinsically resistant.

4.1 Monitoring of antimicrobial resistance

IntroductionThis monitoring programme started in 1998. It is coordinated by

ID-Lelystad and carried out in cooperation with the RIVM.

The programme is aimed at potential zoonotic aspects of antimicrobial

resistance in animals by transfer of resistant strains or resistance genes

from animals to humans. Resistance can be transferred from animals to

humans in several ways. The most important transfer route is considered

to be the food chain. Direct contact may be important for specific animal

owners but is not considered to be a general public health risk. For this

reason the major food animal species (broilers, slaughter pigs and veal

calves) are included in the programme and pets are excluded.

Because of the zoonotic aspect, the bacterial species involved are

zoonotic food-borne pathogens Salmonella spp. and Campylobacter spp..

Moreover, indicator organisms for the normal gut flora are included.

Escherichia coli as indicator for the Gram-negative flora and

Enterococcus faecium as indicator for the Gram-positive flora.

The purposes of this monitoring programme are to detect the emergence

of new resistant phenotypes, determ trends in resistance in time,

and detect potential public health risks.

Materials and methods

Bacterial strains

The indicator organisms E. coli and E. faecium were isolated from faecal

samples from healthy animals at slaughter, for which six pig and six

broiler slaughterhouses, respectively, were randomly selected. These

slaughterhouses were situated all over the country to eliminate potential

regional differences. The sampling period was from July to December

1998. In 1999, sampling began in November to allow detection of the

potential effect of the ban on bacitracin, tylosin, spiramycin and virginia-

mycin, introduced on 1 July 1999, on resistance proportions. Once a day,

at each slaughterhouse, a faecal sample was taken aseptically from one

animal (from one group or flock). Samples from broilers were collected

from the caeca. The vials were stored at –20°C until the next Monday,

when they were sent to ID-Lelystad. At the Department of Bacteriology

the samples were directly 1:10 (w/v) suspended in buffered peptone

solution with 20% glycerol and stored at –20°C pending analysis.

Both human and animal strains of Salmonella spp. were isolated.

All human strains were clinical isolates sent to RIVM for sero- and phage-

typing. The strains isolated in 1998 and 1999 consisted of the following

serotypes: 154 S. Typhimurium (59 times DT 104), 84 S. Enteritidis and

12 S. Typhi. Strains isolated in 2000 consisted of the following serotypes:

501 S. Typhimurium (51 times DT 104 and 26 times DT 204), 208 S. Enteritidis

(103 times Pt 4), and 3 S. Typhi. The other strains were different serotypes.


Chapter 4

Antimicrobial Resistance

• Bacitracin resistance in 1998 was lowest in pigs (83%) compared with

calves and broilers (( 99%). Bacitracin is licensed for use in slaughter

pigs up to six months of age. Salinomycin and tylosin were probably

the AMGPs used most in pigs.

• The proportions of strains resistant to quinupristin/dalfopristin

(Synercid) were always much higher than those resistant to virginia-

mycin. The breakpoint used (2 µg/ml) was probably one dilution step

too low. Using breakpoint 4 µg/ml would result in similar resistance

proportions for virginiamycin and Synercid, which are cross-

resistant.

E. coli (Table 4.1.2)

• Ciprofloxacin resistance (breakpoint 2 µg/ml) was not observed in

pigs. In broilers and veal calves the levels are ( 5%. Flumequin is a

better indicator for decreased quinolone susceptibility because the

breakpoint used (4 µg/ml) is close to the susceptibility of the normal

population. High resistance proportions in broilers and to a lesser

extend in veal calves reflect use practices of (fluoro)quinolones in

these animal species. In pigs (fluoro)quinolones are hardly used.

• Twenty-five human clinical isolates of E. coli O157 (1998) were also

tested for their susceptibility. All strains except one were susceptible

to all antibiotics. The one strain showed the resistance phenotype

similar to that of S. Typhimurium DT104. The strain was resistant to

amoxicillin/piperacillin, doxycycline, trimethoprim, TmpS and

chloramphenicol. This indicates transfer of linked resistance genes

on an integron from either E. coli or other Gram-negative bacteria to

O157.

Salmonella spp. (Tables 4.1.3 and 4.1.4)

• Resistance proportions were slightly higher in human strains than in

strains from food animals (i.e. amoxicillin, doxycycline, chlorampheni-

col, florfenicol). The reason is unclear. Most human strains were of

animal origin and perhaps resistance is a factor involved in pathogeni-

city. Numbers of DT104 (80% pentaresistant) in the human population

were much higher than in animals and this may have affected the

results. In September 2000 26 human clinical isolates of S. Typhimuri-

um 204, resistant to amoxicillin, gentamicin, doxycycline, trimethoprim,

trimethoprim/sulfamethoxazole, chloramphenicol, flumequin and

neomycin have affected the overall results as well. In 2000 79 strains

were resistant to flumequin, but the MIC of ciprofloxacin was still just

below the breakpoint (2 µg/ml). The 53 human flumequin resistant

strains consisted of various serotypes, including 26 S. Typhimurium DT

204 and 16 S. Enteritidis of various phage types. The 26 flumequin

resistant animal strains originated mainly from poultry (25) and one

was isolated in a slaughter pig. The poultry strains mainly consisted of

S. Virchov (22).

Campylobacter spp. (Table 4.1.5)

• MIC values have been determined by micro broth dilution. A NCCLS

standard is being developed at the moment and no validated infor-

mation was available on control strains. Resistance is determined

phenotypically, based on MIC distributions of the populations

examined and NCCLS breakpoints.

• Acquired resistance is quite common, both in human and animal

strains, for erythromycin, doxycycline, amoxicillin and gentamicin

(not detected in 6 pig strains). Resistance proportions for ciprofloxa-

cin are high, but the methodology may have affected the levels and

their validity is not entirely certain.

• Resistance to metronidazole was determined by an e-test. The results

are reliable. High resistance proportions have been described in the

literature as well.

Conclusions

• (Multiple) acquired resistance is very common in E. faecium and

E. coli as commensal organisms in the gut flora isolated from food

animals. The resistance proportions reflect the effect of the use of

antibiotics and AMGPs in the animals.

• Resistance in Salmonella spp. isolated from healthy animals is low.

• A positive effect of a ban on AMGPs on proportions of resistance was

observed for vancomycin (avoparcin), macrolides and virginiamycin.

• No effect was observed of the ban on zinc bacitracin.

• The increased use of avilamycin in broilers resulted in higher

proportions of resistance after the ban on the other AMGPs.

• Acquired resistance in Campylobacter spp. is very common both in

human and in animal strains.


49

Table 4.1.1 Resistance percentage of E. faecium isolated from food animals

Slaughter pigs Broilers Veal calves

1998 1999 1998 1999 1996–1997

Antibiotics N R (%) N R (%) N R (%) N R (%) N R (%)

Amoxicillin 310 0 158 0 314 1 223 2 105 0

Amox/clavulanic acid 310 0.3 158 0 314 2 223 2 105 0

Chloramphenicol 310 0.3 158 0 314 1 223 0 105 0

Doxycycline 310 93 158 75 314 84 223 76 105 96

Erythromycin 310 82 158 57 314 75 223 61 105 86

Vancomycin 310 6 158 8 314 2 223 2 105 42

Teicoplanin 310 6 158 8 314 2 223 2 105 39

Strep > 2000 310 29 158 10 314 9 223 7 105 16

Genta > 500 310 0 158 0 314 0.6 223 2 105 1

Ciprofloxacin 310 5 158 6 314 57 223 46 105 60

Nitrofurantoin 310 - 158 - 314 - 223 - 105 -

Avilamycin 310 0.3 158 1 314 24 223 67 105 0

Bacitracin 310 83 158 82 314 99 223 98 105 100

Flavomycin 310 100 158 100 314 95 223 96 105 97

Salinomycin 310 23 158 35 314 74 223 53 105 0

Quinu/dalfopristin 310 55 158 28 314 71 223 53 105 70

Tilmicosin 310 82 158 56 314 74 223 57 105 86

Virginiamycin 310 36 158 16 314 63 223 46 105 65

Everninomycin 244 1 ND 273 22 ND 24 0


Table 4.1.2 Resistance percentage of E. coli isolated from food animals

Slaughter pigs Broilers Veal calves

1998 1999 1998 1999 1996–1998

Antibiotics N R (%) N R (%) N R (%) N R (%) N R (%)

Amoxicillin 318 14 302 16 318 36 303 38 291 36

Amox/clavulanic acid 317 0 302 2 317 1 301 3 291 5

Piperacillin 317 14 302 16 318 36 303 38 291 36

Cefotaxim 317 0 302 0 318 1 303 2 291 2

Ceftazidim 317 0 302 0.3 318 1 302 2 291 3

Imipenem 317 0 302 2 318 0 303 1 291 0

Gentamicin 318 0.3 302 0 318 2 303 3 291 1

Doxycycline 318 53 302 48 318 64 303 55 291 87

Trmethoprim 318 33 302 33 318 47 303 36 291 45

TmpS 318 31 302 32 318 38 303 36 291 43

Ciprofloxacin 318 0 299 0 317 3 303 2 291 5

Nitrofurantoin 318 - 302 - 318 - 303 - 291 -

Chloramphenicol 318 12 302 8 318 6 303 5 291 36

Florfenicol 318 0 302 0 318 0.3 303 1 291 2

Carbadox 318 0 302 0.3 318 0 303 1 291 0.3

Flumequin 318 2 302 0.3 318 35 303 40 291 16

50

Table 4.1.3 Resistance percentage of Salmonella spp. isolated from humans and food animals

Humans Food animals

1998/1999 2000 1998/1999 2000

Antibiotics N R (%) N R (%) N R (%) N R (%)

Amoxicillin 696 15 359 16 209 7 440 13

Amox/clavulanic acid 696 4 92 4 209 0.4 124 10

Piperacillin 696 15 92 13 209 7 124 15

Cefotaxim 696 1 359 0 209 0 440 0

Ceftazidim 696 1 92 0 209 0 124 0

Imipenem 696 0.1 338 0.9 209 0 425 0.2

Gentamicin 696 0.1 359 7 209 5 440 0.2

Doxycycline 696 23 359 22 209 9 440 25

Trmethoprim 696 24 358 12 205 23 440 19

TmpS 696 6 358 12 209 10 440 19

Ciprofloxacin 696 0 358 0 209 0 440 0

Nitrofurantoin 696 - 92 - 209 - 124 -

Chloramphenicol 696 9 359 12 209 1 440 8

Florfenicol 696 8 359 3 209 0 440 6

Carbadox 696 0 92 1 209 0 124 0

Flumequin 696 3 359 15 209 2 440 6

Cefuroxime - - 267 0 - - 316 0

Neomycin - - 267 9 - - 316 0.3

Table 4.1.4 Resistance percentage of Salmonella spp. for each animal species

Pigs Cattle

1998/1999 2000 1998/1999 2000


Amoxicillin 31 6 153 16 18 0 26 58

Amox/clavulanic acid 28 0 54 6 18 0 15 40

Piperacillin 32 6 54 16 18 0 15 67

Cefotaxim 26 0 153 0 18 0 26 0

Ceftazidim 26 0 54 0 18 0 15 0

Imipenem 26 0 146 0 18 0 26 0

Gentamicin 27 0 153 0 18 0 26 4

Doxycycline 32 19 153 23 18 11 26 58

Trmethoprim 29 31 153 16 18 22 26 12

TmpS 25 2 153 16 18 0 26 12

Ciprofloxacin 26 0 153 0 18 0 26 0

Nitrofurantoin 32 - 54 - 18 - 15 -


Florfenicol 32 0 153 13 18 0 26 50

Carbadox 32 0 54 0 18 0 15 0

Flumequin 345 0 153 0 18 0 26 0

Cefuroxime - - 99 0 - - 11 0

Neomycin - - 99 0 - - 11 0


51

Table 4.1.4 Resistance percentage of Salmonella spp. for each animal species continue

Broilers Laying hens

1998/1999 2000 1998/1999 2000


Amoxicillin 69 17 86 22 94 0 93 3

Amox/clavulanic acid 69 0 23 0 94 0 28 7

Piperacillin 69 17 23 0 94 0 28 0

Cefotaxim 69 0 86 0 94 0 93 0

Ceftazidim 69 0 23 0 94 0 28 0

Imipenem 69 0 79 0 94 0 92 1

Gentamicin 69 10 86 0 94 3 93 0

Doxycycline 69 10 86 13 94 3 93 5

Trmethoprim 69 25 86 52 91 19 93 0

TmpS 69 16 86 52 94 4 93 0

Ciprofloxacin 69 0 86 0 94 0 93 0

Nitrofurantoin 69 - 23 - 94 - 28 -


Florfenicol 69 0 86 0 94 0 93 0

Carbadox 69 0 23 0 94 0 28 0

Flumequin 69 7 86 22 94 0 93 2

Cefuroxime - - 63 0 - - 65 0

Neomycin - - 63 0 - - 65 0

Table 4.1.5 Resistance percentage of Campylobacter spp. isolated from humans and food animals

Humans Pigs Broilers Cattle

1998 1998 1999 1999


Erythromycin 76 21 6 0 21 33 32 44

Doxycycline 77 12 6 0 21 19 32 34

Gentamicin 77 9 6 0 21 19 32 28

Carbadox 63 ? 6 ? 21 ? 32 ?

Flavomycin 77 ? 6 ? 21 ? 32 ?

Ciprofloxacin 77 16 6 83 21 14 32 34

TmpS 77 0 6 0 21 0 32 0

Amoxicillin 77 52 6 0 21 43 32 34

Metronidazole 44 52 - ND 9 67 13 54


4.2 Vancomycin-resistant enterococci (VRE)

IntroductionDuring the past 20 years an increase in VRE has been observed in hospi-

tals, mainly in the United States, while VRE seemed to be virtually absent

in the community. This is in contrast to Europe, where VRE can easily be

detected outside hospitals in non-hospitalized persons and farm animals.

The high prevalence of VRE in farm animals in Europe is thought to be the

result of the use of the glycopeptide antibiotic avoparcin as an antimicro-

bial growth promoter. Consequently, VRE from animal husbandry may

enter the food chain and subsequently spread to humans.

The most prevalent vancomycin resistance transposon is Tn1546, which

confers high-level transferable resistance to vancomycin, teicoplanin and

avoparcin. Whether an animal reservoir of VRE actually poses a threat to

humans depends on the level of exposure of humans to animal strains,

on the ability of animal strains to colonize the human gut for a prolonged

time and on the frequencies of horizontal transmission of Tn1546 in the

case of transient colonization of humans by animal-derived VRE.

Genetic relationship of VRE from various human and animal sources

Amplified-fragment length polymorphism (AFLP) was used to investigate

the genetic relationships between 357 vancomycin-resistant

Enterococcus faecium (VREF) strains isolated from hospitalized patients,

non-hospitalized persons and various animal sources in the Netherlands.

The isolates from non-hospitalized persons (n = 31), pigs (n = 110), poultry

(n = 34) and veal calves (n = 48) were all epidemiological unrelated, while

the isolates from hospitalized patients (n = 134) were either single isolates

originating from a survey in nine Dutch hospitals (n = 11) or from three

hospital outbreaks in Amsterdam (n = 33), Amersfoort (n = 41) and Utrecht

(n = 49).

AFLP-typing revealed four major genogroups (A-D) which were associa-

ted with particular hosts and environments (Fig. 4.2.1 ). All strains from

pigs (110) were found in genogroup A, while the majority of poultry

isolates (32/34) and veal calves isolates (39/48) clustered in genogroups B

and D respectively (Fig. 4.2.1). VREF isolates from non-hospitalized

persons and hospitalized patients clustered differently. Strains from non-

hospitalized persons were predominantly found in genogroup A (29/31)

together with all the pig isolates, while the isolates from hospitalized

persons were found in genogroups A (28/134), B (48/134) and C (58/134)

(Table 4.2.1). Although isolates from hospitalized patients were found in

three different genogroups, VREF strains isolated from clinical sites and

strains associated with one of the three hospital outbreaks were only

found in genogroups B and C which indicates that VREF strains with

clinical importance are genetically different from VREF strains present in

the gut of non-hospitalized persons.

Because strains of genotypes D (veal calves) were not found among

humans, these strains may colonize humans only transiently. In contrast,

all isolates from pigs clustered together in genogroup A with strains from

non-hospitalized persons, suggesting that, in the community, VREF strains

in humans may originate from pigs. Furthermore, strains from poultry in

genogroup B grouped with approximately a third of the strains from

hospitalized patients, suggesting that at least part of the clinically

relevant VREF isolates may originate from poultry.

Transmission of vancomycin-resistance genes

Molecular analysis of the vanA-containing transposon Tn1546 revealed

the existence of numerous Tn1546 variants. The differences between

Tn1546 variants included point mutations, insertions of insertion sequence

(IS) elements and deletion often associated with IS-element insertions.

In general, identical Tn1546 variants were found among human- and

animal-derived VRE. The distribution of four Tn1546 subtypes, A1, A2, A3,

and E, among a set of 295 different human and animal sources in the

Netherlands was studied in more detail (Table 4.2.1).

The most predominant transposon type among isolates recovered from

humans and pigs was type A2. This type was found in 99% of the pig

isolates, in 75% of the isolates from non-hospitalized persons and in 88%

of the isolates from hospitalized patients. This suggests an epidemiolo-

gical link between pig and human reservoir and confirms the results of

the AFLP analysis. Furthermore, type A2 was found in 16% of the veal calf

isolates and in 3% of the isolates recovered from poultry.

Transposon type A1 is also recovered from different human and animal

isolates. The fact that the transposon types A1 and A2 were found among

genetically different VREF strains indicates horizontal transfer of these

transposon types.

In contrast, transposon types A3 and E displayed a more restricted

distribution. Type A3 was exclusively found among VREF recovered from

veal calves and type E was predominantly found in VREF recovered from

poultry. This may indicate that Tn1546 variants from these animal sources

are rarely transferred to humans.

Conclusions

VRE strains are predominantly host-specific. Strains present in the gut of

non-hospitalized individuals are similar to strains recovered from pigs, but

genetically different from the prevailing strains isolated from hospitalized

patients.

Identical vanA transposons were found in strains recovered from non-

hospitalized persons, hospitalized patients, pigs, poultry, and veal calves,

suggesting horizontal spread of the vanA transposon among genetically

different enterococci.


Table 4.2.1 Distribution of Tn1546 types among VREF recovered

from various human and animal sources

Source A1 A2 A3 E Number

Non-hospitalized 22% 75% 0 3% 32

Hospitalized 7% 88% 0 5% 41

Pigs 1% 99% 0 0 125

Poultry 44% 3% 0 53% 36

Veal calves 20% 16% 64% 0 6

VRE carriage in outpatients before and after theavoparcin ban

Introduction

A study was carried out to investigate whether the ban on the use of

avoparcin as antimicrobial growth promoter, implemented in April 1997,

affected the prevalence of VRE colonization in non-hospitalized persons.

Investigation and results

Forty-four general practitioners collected faeces samples from a random

selection of patients consulting their practice. Samples taken in the

period October 1996 to April 1997, before the ban, and from April 1998 to

May 1999, after the ban, were analysed for the presence of VRE using

selective enrichment media.

The number of faecal samples containing high-level resistant vanA

Enterococcus faecium isolates taken after the ban was lower than in the

samples taken before the ban (Table 4.2.2) This difference is marginally

significant (p = 0.06). No differences were seen in the number of samples

containing low-level resistant Enterococcus gallinarum or Enterococcus

flavescens/casseliflavus.

Conclusion

These results strongly suggest that the discontinuation of the use of

avoparcin resulted in a decreased colonization rate and may finally result

in eradication of high-level resistant VRE colonization in non-hospitalized

individuals.


Table 4.2.2 Prevalence of VRE in human faecal samples

collected in general practices

Species Before the ban After the ban

N=124 % N=167 %

E. faecium (vanA) 10 8% 5 3

E. gallinarum 8 6% 11 7

E. lavescens/casseliflavus 2 2% 1 0.6

Total VRE 20 16% 17 10


Genogroup A

Genogroup B

Genogroup C

Genogroup D

Figure 4.2.1 Abridged dendrogram showing the genetic relationships of 357 vancomycin-resistant Enterococcus faecium strains originating

from different human and animal sources. Numbers on the horizontal axe indicate % similarities

Source number (% of source)

Pigs 110 (100%)

Nonhosp. persons 29 (94%)

Hosp. patients 28 (21%)

Poultry 2 (6%)

Source number % of source

hosp. Patients 48 36%

(Amsterdam outbreak)

poultry 32 94%

veal calves 9 19%

nonhosp. Persons 2 6%


hosp. patients 58 (43%)

(Amersfoort and

Utrecht outbreak)


veal calves 39 (81%)

The prevalence of resistance (%) in the population for a certain antibiotic

is calculated as the number of samples showing growth of E. coli or

enterococci on the plates containing that antibiotic, divided by the total

numbers of samples tested x 100%.

The degree of resistance (%) of each faecal sample to each of the

antimicrobial agents tested was calculated as the number of bacterial

colonies growing on the plate containing that agent divided by the total

number of typical colonies on the antibiotic free control plate x 100%.

Two degrees of antibiotic resistance to a particular antibiotic could be

distinguished: low degree of resistance, i.e. less than 50% of the total

number of the indicator microorganisms present per gram faeces was

resistant, and high degree of resistance, i.e. 50% or more (thus the

majority) of the indicator microorganisms resistant to that agent.

The prevalence of a high degree (%) is the number of samples with a high

degree of resistance to a particular antibiotic divided by the total number

of samples tested x 100%.

4.3 Antibiotic resistance in food animals andassociated public health risks

Introduction

The possibility and the risks of transfer of resistant bacteria and/or

resistance genes from food animals to man has been studied by the

department of medical microbiology of the university of Maastricht in

cooperation with the RIVM as part of a larger study of antibiotic

resistance, methods for quantifying it and the effects of interventions in

the open population, i.e. healthy Dutch individuals outside hospitals.

The possibility and risks of transfer are studied by determining the

prevalence of the degree of resistance of indicator bacteria, i.e.

Escherichia coli and enterococci in the faecal flora of food animals and in

populations of humans with different degrees of contact with these

animals: farmers in direct daily contact with their animals and excreted

faeces, slaughterers in daily contact with animal carcasses, and

(sub)urban residents who are only indirectly exposed to the faecal flora of

food animals via foods of animal origin. Faecal samples are diluted and

directly plated on selective antibiotic containing agar plates, as described

previously. This method is more sensitive than selecting at random a single

isolate and determining its resistance.



Study I: Prevalence and degree of antibiotic resistance in faecal samples of pigs collected at slaughter in five Dutch abattoirs and in

Sweden

The prevalence and degree of antibiotic resistance of the faecal indicator bacteria Escherichia coli and enterococci were

determined in faecal samples of pigs collected at five Dutch abattoirs and in samples of Swedish pigs. As shown in Table 4.3.1

and 4.3.2, in the Dutch pig populations the prevalence and degree of resistance both of E. coli and enterococci were high for

antibiotics regularly used in pig medicine or as antimicrobial growth promoters (AMGPs). Differences were observed

between the five abattoirs, but could not be explained as no data on antibiotic consumption by the different pig populations

could be obtained. In the Swedish samples the resistance of enterococci against antibiotics used as AMGPs was significantly

lower than in the Dutch populations and avoparcin resistance was even absent. This was to be expected as Sweden had

already banned the use of AMGP in 1986. Therefore, such a ban seems to be effective in lowering the prevalence of

resistance in enterococci in the faecal flora of pigs. The overall lower prevalence and degree of resistance against antibiotics

used therapeutically observed in the Swedish pig samples was similar to that in the Dutch pig populations. This indicates that

banning the use of antibiotics as AMGPs does not inevitably cause a higher therapeutic use of antibiotics in pigs. Moreover,

comparison of the results from the Swedish and Dutch samples indicate that monitoring the prevalence and degree of

resistance in faecal samples collected at slaughterhouses can be used to detect differences in antibiotic consumption

between different pig populations.

Table 4.3.1 Prevalence of antibiotic-resistant Escherichia coli

and percentage of samples with a high degree (HD)

of resistance in pig faecal samples collected at five

abattoirs in the Netherlands and Sweden

Antibiotic Slaughterhouses

Concentration in agar Netherlands Sweden

(n=1320)1 (n=100)

(mg/l) Prev. HD Prev. HD

Amoxycillin 25 85* 13** 51 3

Oxytetracycline 25 93* 40* 69 6

Chloramphenicol 25 63*2 3 3 0

Nitrofurantoin 50 3 0 0 0

Trimethoprim 8 85* 21* 46 1

Neomycine 32 56* 4 17 3

Getamicin 16 2 0 0 0

Flumequin 16 3 0 1 0

Ciprofloxacin 4 1 0 0 0

1 number of samples tested * significantly different from Sweden p < 0.0012 n = 1022 ** significantly different from Sweden p = 0.005

Table 4.3.2 Prevalence of antibiotic-resistant enterococci and

percentage of samples with a high degree (HD) of

resistance in pig faecal samples collected at five

abattoirs in the Netherlands and Sweden

Antibiotic Slaughterhouses

Concentration in agar Netherlands Sweden

(n=1320)1 (n=100)

(mg/l) Prev. HD Prev. HD

Amoxycillin 25 0 0 0 0

Gentamicin 500 33 0 0 0

Oxytetracycline 25 93* 3 46* 67 6

Erythromycin 10 95* 70* 63 3

Vancomycin 10 39* 2 0 0

Dalfopristin-

Quinupristin 8 72* NT2 45 NT2

1 number of samples tested * significantly different from Sweden p < 0.0012 not tested3 n = 1022


Study II: Vancomycin-resistant enterococci in turkey flocks, turkey farmers and urban residents in the Netherlands; indications for

clonal and genetic dissemination of vancomycine resistance from turkeys to man

Another study determined the prevalence and degree of resistance against antibiotics commonly used for therapy or as

AMGP in poultry in the faecal flora of turkeys and three human populations, each with a different level of contact with turkeys:

turkey farmers, turkey slaughterers and (sub)urban residents. The prevalence of vancomycin-resistant enterococci (VRE) was

significantly higher (62%) in turkeys fed avoparcin than in turkeys not exposed to this drug (8%). No significant differences

between farmers using avoparcin (37%) and those who did not (42%) were observed. The prevalence in turkey slaughterers

and (sub)urban residents was 20% and 13% respectively. The pulsed field gel electrophoresis (PFGE) patterns of the isolated

VRE in the different populations were quite heterogeneous, but isolates with the same pattern (similar genotypes) were found

among turkey and turkey farmer isolates. On two occasions Enterococcus faecium isolates with identical PFGE patterns were

found in the faeces from the farmer and turkeys of the same farm, which in addition contained an indistinguishable vanA-

containing element not found in human isolates before. This strongly suggested transfer of resistant enterococci from animals

to humans. Moreover similar vanA elements were not only found in isolates with the same PFGE patterns, but also in other

strains isolated from humans and turkeys, which indicated that resistant strains spread not only clonally, but also via a

spillover of resistance genes of turkey enterococci to enterococci in the human intestinal flora.

Study III: Faecal carriage of antibiotic-resistant enterococci by chicken and chicken farmers and slaughterers in the Netherlands;

clonal and genetic dissemination of vancomycine resistance from poultry to man

Similar observations were made by determining the prevalence and degree of antibiotic resistance of enterococci in the

faecal flora of two chicken populations, i.e. laying hens with a low exposure and broilers with a high exposure to antibiotics,

and likewise in faecal samples from laying hen farmers, broiler farmers and poultry slaughterers. The prevalence and degree

of resistance for most antibiotics tested was higher in broilers than in laying hens, and in broiler farmers and broiler

slaughterers than in laying hen farmers. Hence a clear correlation was found between the prevalence and degree of

resistance found in faecal enterococci and the amounts of antibiotics the chicken populations were exposed to. Among the

human populations there was no difference in antibiotic consumption, and the observed prevalence and degree of resistance

correlated with these parameters in the animals they were in contact with. The overall prevalence of resistance of faecal

enterococci was significantly higher in broilers and broiler slaughterers than in laying hen farmers. The PFGE patterns of the

isolated VRE (one per VRE positive faecal sample) of the five populations were quite heterogeneous, but in two cases

Enterococcus hirae with identical PFGE patterns were isolated from the farmer and from broilers from the same farm.

Moreover, similar vanA-elements were not only found in isolates with the same PFGE-patterns but also in other VRE isolated

from humans and chickens. These results again strongly indicated transmission of resistant enterococci from poultry to man,

both by clonal spread and by transposon transfer from animal strains to enterococci in the human intestinal flora.


Table 4.3.3 Prevalence (%) of resistant faecal E. coli in different

populations in 1997

Population n Ciprofloxacin Tetracycline

Turkeys and broilers 97 47 82

Laying hens 25 0 76

Pigs 291 2 100

Turkeys and

broiler farmers 98 15 70

Laying hen farmers 25 0 36

Pig farmers 290 1 79

Urban residents 117 <1 31

Study IV: Faecal carriage of antibiotic resistant Escherichia coli by poultry and poultry farmers and slaughterers in the Netherlands;

clonal dissemination of fluoroquinolone (Baytril™) resistant E. coli from poultry to man

The prevalence and degree of resistance of E. coli were determined in faecal samples of three poultry populations: broilers

and turkeys commonly exposed to antibiotics and laying hens with a low usage of antibiotics. In addition faecal samples from

five human populations were examined: turkey and broiler farmers, laying hen farmers and broiler slaughterers and turkey

slaughterers. The antibiotics tested or analogues are commonly used in poultry medicine. Ciprofloxacin-resistant isolates

from these 8 populations and from poultry meat samples were genotyped by PFGE. The prevalence and degree of resistance

were significantly higher in turkeys and broilers than in the laying hen population. Also, for most antibiotics the observed

resistance in faecal E. coli of turkey and broiler farmers, and of turkey and broiler slaughterers, was higher than in laying hen

farmers. The same resistance patterns were found in turkey, turkey farmer and turkey slaughterer isolates, and in broiler,

broiler farmer and broiler slaughterer isolates. Multiresistant isolates were common in turkey and broiler farmers but absent

in the laying hen farmer population. The prevalence of fluoroquinolone resistance in poultry and poultry farmers was

compared with those observed in a former study on pigs and pig farmers (Table 4.3.3). The fact that fluoroquinolones are not

commonly used in pigs and laying adult hens was clearly reflected in the prevalence of fluoroquinolone-resistant E. coli in

these populations. The higher prevalence observed in broiler and turkey farmers than in the other studied human populations

could only be explained by dissemination from the animals to their respective farmers. PFGE patterns of the fluoroquinolone-

resistant isolates from the eight populations were quite heterogeneous, but on two occasions E. coli strains with an identical

PFGE pattern were isolated from turkeys and the farmer from the same farm, and also from a broiler and a broiler farmer,

but from two different farms. Moreover, three E. coli isolates from turkey meat were identical to faecal isolates from turkeys.

The results of this study strongly suggest that transmission of resistant clones of E. coli and resistance plasmids from poultry

strains to human E. coli does commonly occur.

59

Table 4.3.4 Prevalence of antibiotic resistant enterococci and percentage of samples with a high degree (HD) of resistance in faecal samples

collected in the south of the Netherlands

Antibiotic 1997 1999

Concentration in agar Humans Pigs Broilers Humans Pigs Broilers

(n = 117)1 (n = 282) (n = 50) (n = 171) (n = 127) (n = 89)

(mg/l) Prev. HD Prev. HD Prev. HD Prev. HD Prev. HD Prev. HD

Vancomycin 10 12 0 34 3 80 8 6* 0 17** 0 31** 0

Erythromycin 10 50 11 84 67 94 44 47 8 85 65 92 20

Dalfopristin-

Quinupristin 8 30 NT2 75 NT 92* NT 12** NT 31** NT 57** NT


Conclusions

A. Resistant bacteria are disseminated from food animals to humans and

not only colonize humans, but also exchange their resistance genes

with bacteria of the human intestinal flora.

B. The higher the prevalence and degree of resistance in the intestinal

flora, the higher the risk of transfer to the human population.

Therefore, a low prevalence of resistance in the intestinal flora of

food animals should be considered as a safety mark and public health

goal.

1 number of samples tested * significantly different p < 0.0512 not tested ** significantly different p < 0.001

Study V: Banning antimicrobial growth promoters in animal feeds has positive public health effects; intervention is effective

The effect of the suspension of avoparcin usage as AMGP in the Netherlands in 1997 was assessed. The prevalence and

degree of enterococci resistant to three antibiotics important for human therapy, and closely related to and cross-resistant

with three commonly used AMGP, were analysed in faecal samples of pigs and broilers collected at the same slaughter-

houses in 1997 and 1999, and from healthy human (sub)urban residents from the same cities and collected in the same years.

The prevalence of VRE decreased significantly in all three populations (Table 4.3.4). Erythromycin resistance did not change,

but dalfopristin-quinupristin resistance had declined, despite the fact that the use of the related AMGP, i.e. virginiamycin,

was not banned at that time. Virginiamycin, however, was in short supply in the Netherlands in 1998 and 1999 because of

production problems. These results indicate that continuation of the ban in the Netherlands on the use of avoparcin in animal

feeds will not only eradicate VRE from food animals but also from the human population. Because of cross-resistance with

macrolides and lincosamides, which are both commonly used in human and veterinary medicine, complete disappearance of

pristinamycin resistance after a ban of virginiamycin is less likely to occur. The use of vancomycin in humans had not changed

between 1997 and 1999. Therefore, the results of this study again show that use of antibiotics in food animals not only causes

resistance in the intestinal flora of these animals but that these resistant bacteria or their resistance genes are transferred to

humans via the food chain.

C. A low prevalence and degree of resistance in the faecal flora of

animals can be achieved by using less antibiotics in these animals.

Stopping the use of the antimicrobial growth promoters results not

only in a reduced prevalence and degree of resistance in the faecal

flora of exposed food animal populations, but also in human

populations consuming foods from these animals.

D. Banning the use of antimicrobial growth promoters does not increase

the need for greater therapeutic use of antibiotics.


Zoonoses and zoonotic agents in humans, food, anim

als and feed in the Netherlands, 2001

Zoonoses and zoonotic agents in humans, food,

animals and feed in the Netherlands 2001 is

published by the Inspectorate for Health

Protection and Veterary Public Health

(Keuringsdienst van Waren) in close

collaboration with the National Institute

for Public Health and Environment (RIVM).

Collaborating institutes

KvW - Inspectorate for Health Protection

and Veterinary Public Health

P.O. Box 16108

2500 BC The Hague

tel 0800-0488 or +31 (0)70 3407001

fax +31 (0)70 3407080

www.keuringsdienstvanwaren.nl

RIVM - National Institute for Public Health

and the Environment

Antonie van Leeuwenhoeklaan 9

P.O. Box 1

3720 BA Bilthoven

ID-Lelystad - Institute for Animal Science and Health

P.O. Box 65

8200 AB Lelystad

GD - Animal Health Service

P.O. Box 9

7400 AA Deventer

VVDO - Department of the Science of Food

of Animal Origin

P.O. Box 80163

3508 TD Utrecht

RVV - National Inspection Service for

Livestock and Meat

Burg. Feithplein 1

Postbus 3000

2270 JA Voorburg

EUR - Erasmus University Rotterdam

Institute of virology

P.O. Box 1738

3000 DR Rotterdam

PVE - Production Boards for Livestock,

Meat and Eggs

P.O. Box 5805

2280 HV Rijswijk

University Hospital Maastricht

Departement of Medical Microbiology

P.O. Box 5800

6202 AZ Maastricht

Zoonoses and zoonotic agents in humans, food, animalsand feed in the Netherlands2001

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