the effect of different housing systems on salmonella and antimicrobial resistance in laying hens
TRANSCRIPT
THE EFFECT OF DIFFERENT HOUSING SYSTEMS ON
SALMONELLA AND ANTIMICROBIAL RESISTANCE
IN LAYING HENS
SEBASTIAAN VAN HOOREBEKE
Thesis submitted in fulfillment of the requirements for the degree of Doctor in Veterinary
Sciences (PhD), Faculty of Veterinary Medicine, Ghent University, 2010
Promoters:
Prof. Dr. Jeroen Dewulf
Prof. Dr. Filip Van Immerseel
Faculty of Veterinary Medicine
Department of Reproduction, Obstetrics and Herd Health
This research was funded by the EU FP6, under the contract 035547 (Safehouse project)
Schilderij kaft: Koen Pattyn
Printed by: Ryhove Plot-it
THE EFFECT OF DIFFERENT HOUSING SYSTEMS ON
SALMONELLA AND ANTIMICROBIAL RESISTANCE
IN LAYING HENS
SEBASTIAAN VAN HOOREBEKE
Thesis submitted in fulfillment of the requirements for the degree of Doctor in Veterinary
Sciences (PhD), Faculty of Veterinary Medicine, Ghent University, 2010
Promoters:
Prof. Dr. Jeroen Dewulf
Prof. Dr. Filip Van Immerseel
Faculty of Veterinary Medicine
Department of Reproduction, Obstetrics and Herd Health
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
CHAPTER 1 General introduction 1
1. Laying hen husbandry 4
2. The role of eggs in human salmonellosis 13
3. Epidemiology of Salmonella 21
4. Antimicrobial resistance in laying hens 32
5. References 36
CHAPTER 2 Scientific aims of the thesis 55
CHAPTER 3 Faecal sampling under-estimates the actual prevalence of Salmonella in laying
hen flocks 59
CHAPTER 4 The age of a production system and previous Salmonella infections on farm
are risk factors for low-level Salmonella infections in vaccinated laying hen
flocks 73
CHAPTER 5 Determination of the within and between flock prevalence and identification of
risk factors for Salmonella shedding in laying hen flocks housed in
conventional and alternative systems 89
CHAPTER 6 The effect of the housing system on antimicrobial resistance in indicator
bacteria in laying hens 109
CHAPTER 7 General discussion 135
SUMMARY 161
SAMENVATTING 167
CURRICULUM VITAE
DANKWOORD
BIBLIOGRAPHY
LIST OF ABBREVIATIONS
BGA brilliant green agar
BPW buffered peptone water
CI confidence interval
CLSI Clinical and Laboratory Standards Institute
DANMAP Danish Integrated Antimicrobial Resistance Monitoring and Research Program
EFSA European Food Safety Authority
EU European Union
EUCAST European Committee on Antimicrobial Susceptibility Testing
FAVV Federaal Agentschap voor de Veiligheid van de Voedselketen
GEE generalized estimating equitations
MDR
MSRV
multidrug resistance
modified semisolid rappaport vassiliadis agar
OR odds ratio
PT phage type
SPSS Statistical Package for the Social Sciences
subsp. subspecies
VEPEK Vereniging voor Pluimvee, Eieren en Konijnen
XLD xylose lysine decarboxylose
CHAPTER 1
GENERAL INTRODUCTION
Chapter 1 3
PREFACE
Nowadays, consumers have more and more concerns about modern food production
systems, especially with respect to animal welfare, the protection of the environment and food
safety issues. Since these three aspects are strongly linked to each other, implementing
regulations or new standards to improve one aspect may negatively influence other aspects,
creating potential conflicts between regulatory aims (de Passillé and Rushen, 2005).
Perhaps one of the best known manifestations of consumers awareness of animal
welfare is the growing discontent with the conventional industrial egg production, resulting in
an aversion to eggs originating from hens housed in conventional battery cages (Appleby,
2003). This finally lead to the European ban on conventional battery cages (Council Directive
1999/74/EC). From 2012 onwards, laying hens must be housed in enriched cages or non-cage
housing systems such as aviaries, floor-raised and free-range systems.
At the same time food safety has become a very important consumer demand. Eggs
used for human consumption should be free from both microbiological and chemical
contaminants. An important zoonotic pathogen typically associated with eggs and egg
products is Salmonella Enteritidis (Davies and Breslin, 2001; Namata et al., 2008). S.
Enteritidis is, together with S. Typhimurium, the most commonly isolated serotype in human
cases of salmonellosis (WHO, 2006; EFSA, 2007) in Europe.
Another aspect of food safety gaining importance over the last 15 years is the
emergence of antimicrobial resistance in food production animals and the spread of resistant
bacteria from animals to humans. To fully assess the size of the problem in animal production,
standardized and continuous surveillance programs are necessary to monitor the occurrence
and persistence of antimicrobial resistance in food animals (Aarestrup, 2004; Wallmann, 2006;
WHO, 2008). Antimicrobial resistance in pathogenic and indicator bacteria in broilers, pigs
and cattle has been extensively explored and described. However, epidemiological data on the
prevalence of antimicrobial resistance in laying hens is still scarce. Since it has been
described for laying hens that the incidence of bacterial diseases tends to increase in non-cage
systems, possibly resulting in higher antibiotic usage, it is necessary to further explore this
topic.
In this thesis, the influence of different housing systems for laying hens on the
prevalence of Salmonella and antimicrobial resistance in indicator bacteria will be explored.
4 Chapter 1
1. LAYING HEN HUSBANDRY
a. Production chain
Parent breeding flocks
Hatchery
Rearing house
Laying hen house
ProcessingShell eggs for human
consumption
Industry Catering Retail
Hatching eggs
Day-old layer chicks
Pullets
Egg production
Figure 1: Diagram of the egg industry (adapted from Defra, 2004 and VEPEK, 2009)
The egg production chain is characterised by a strongly integrated nature, meaning
there is a strong entanglement between all links of the supply chain. In the flowchart below,
the different stages are schematically represented (Figure 1).
At the top, there are the farms with the parent breeding hens. They produce breeding
eggs that are delivered to the hatcheries. In the hatcheries, day-old layer chicks are produced.
These chicks are transported to the rearing farms. The young pullets stay there till 18 weeks
of age after which they are brought to the egg producing farms. A proportion of the finally
produced eggs is transported to the packing station. From here the eggs go the retail. The
other eggs are not sold as whole table eggs but are processed in the industry and incorporated
Chapter 1 5
in other products, such as sauces, biscuits, frozen fried eggs, pasteurized whole eggs, albumen
and yolks...
Usually, the productive life span of laying hens is around 14 months but depending on
the price of the eggs versus the cost of feed and replacement pullets, the producer can decide
to recycle the current flock by induced moulting to achieve a second, but shorter, laying cycle
(North and Bell, 1990; Holt, 2003). Induced moulting is thought to be economically
advantageous, especially in a market situation with higher egg prices, declining values of hen
carcasses and availability of fewer processors willing to process them (Holt, 2003; Webster,
2003). Feed withdrawal to achieve body weight loss, followed by a rest period and then
restimulation of egg production has since long been the typical industry method for induced
moulting (Webster, 2003), although for welfare reasons this has been forbidden in the EU
(Yousaf and Chaudhry, 2008). Besides welfare issues, it has also been described that the
process of induced moulting has many adverse influences on the presence and shedding of
Salmonella in laying hens (Holt and Porter; 1993; Ricke, 2003; Golden et al., 2007).
Although the egg production chain is very strictly organized from top to bottom, it is
no isolated entity. There are very frequent and intense contacts with other segments of poultry
production in specific and the agricultural sector in general, which implies risks for the spread
of diseases. To illustrate this, the contact structure of the Belgian poultry sector is presented in
Figure 2 with the number of incoming and outgoing contacts per month between the different
segments of the poultry sector.
6 Chapter 1
Figure 2: The contact structure of the Belgian poultry sector (Van Steenwinkel et al., 2010)
b. Housing of laying hens
i. History of the battery cages
For centuries, free-range housing and indoor floor housing were the most common
methods of laying hen husbandry. This changed rapidly when the keeping of laying hens in
cages boomed in the USA during the 1930‟s and 1940‟s. During the following decades this
housing system for laying hens became the most used system worldwide (Bell, 1995; Duncan,
Out Eggs: 1.50/mo. Out Live poultry: 5.49/mo.
Out 0.51/mo.
In 1.75/mo.
In 0.17/mo.
In live poultry: 0.13/mo.
Out Live poultry: 13.54/mo.
Out 7.33/mo.
Out live poultry: 22.78/mo. Out Eggs: 41.67/mo.
Consumption eggs
Out Live poultry: 0.77/mo.
Out 6.00/mo.
In 1.76/mo.
Out 2.07/mo.
Out 3.96/mo.
Lending out
0.99/mo.
Out 1.47/mo.
Live poultry
Out 0.48/mo.
Out 0.30/mo.
In 0.25/mo.
Out 0.08/mo.
Out 0.30/mo.
In 0.50/mo.
Out 0.08/mo.
Hatchery
Rearing Farms
Food industry
Broiler Farms
Multiplier
Show/Wild rearing; Multiple category
farms
Ready to lay hens
Live poultry
Hatching eggs
In 0.08/mo.
Ready to lay hens
One-day-old chicks
Meat Local traders
Private persons
Out 0.60/mo.
Live poultry
Live poultry
Eggs
Out 3.99/mo.
Live poultry
Live poultry
Out 1.68/mo.
Live poultry
Laying Farms Slaughterhouse
Chapter 1 7
2000; Tauson, 2005). Originally, cages were introduced for single laying hens. Later on,
several hens were placed in a cage and group sizes from three to eight hens were typically
used. The term „battery cage‟ is derived from the large number of cages in a laying hen house
which is called a battery of cages (Keeling, 2002; Appleby et al., 2004).
The housing in battery cages had numerous advantages: it gave way to automatization,
it was less labour-intensive for the laying hen keepers and it allowed keeping more hens on
the same surface. Besides these economical and ergonomical advantages it also implied much
higher hygienic standards because the hens were now separated from their own faeces, there
was significant less interference from the environment and there was a reduced risk of
mortality due to cannibalism, feather pecking and aggression (Duncan, 2000; Savory, 2004).
In spite of all these advantages, it is no surprise that from the beginning concerns were
raised about the adverse consequences of battery cage housing for the welfare of the laying
hens, especially because of the very restricted space and the impossibility to perform their
natural behaviour.
ii. The EU ban on battery cages
The European Union took a leading role in the debate on laying hens‟ welfare by
adopting Council Directive 1999/74/EC, stating that from January 1st 2012 onwards the
housing of laying hens in conventional battery cages will be forbidden in all EU member
states. However, it took some time before a consensus on the main points was reached.
The welfare of laying hens was brought to the public attention as early as 1964, when
Ruth Harrison published her book „Animal Machines‟. The book focused principally on the
welfare of animals kept under intensive production systems, such as the battery cages for
laying hens, and it also raised questions about the safety of eating products from animals kept
under such conditions (Fraser, 2001). The public concern, triggered by this book and others,
made that the welfare of farm animals has been under discussion ever since.
To meet this concern, in 1976 the Council of Europe introduced the Convention on the
Protection of Animals kept for farming purposes, with the objective to improve the care,
husbandry and housing of farm animals, especially those in intensive systems (Anon., 1976).
However, the recommendations in the Convention were only described in very general terms.
To counter this, the Council organized a Standing Committee whose task was to work out
some more specific requirements (Appleby, 2003).
8 Chapter 1
The EU was an important player in the process, first by becoming an active party in
the Convention in 1978 and then by financing background scientific work on poultry welfare
in the farm animal welfare coordination program from 1979 (Tarrant, 1983; Appleby, 2003).
All this resulted nearly a decade later in a Directive (Anon., 1986) laying down minimum
standards for the protection of hens in battery cages, being one of the first Europe-wide
statutes that actually specified how animals were to be kept. It stated that by the 1st of January
1988, all newly built battery cages had to provide at least 450 cm2 per hen, and these
standards had to apply to all cages by January 1995. The strength of this Directive was that
EU member states were forced to implement the provisions into their national legislation
(Appleby, 2003).
In 1992, because of ongoing public pressure, a new draft of an EU-Directive was
formulated by the European Commission, although this did not influence the situation in the
field, partially because of the huge scepticism of the egg producing industry. It was not until
March 1998 that the European Commission brought out a new proposal for an EU Directive,
stating that hens should be provided with a nest, litter and perching facilities (Anon., 1998). It
was for the first time that the term „enriched cage‟ was mentioned, defined as „a battery cage
equipped with litter, perches and a „nestbox‟.
Directive 1999/74/EC, laying down minimum standards for the protection of laying
hens, restricts the housing of laying hens in the EU to three different categories: unenriched
cages, enriched cages and alternative systems. The Directive states that the so-called
unenriched cages (i.e. conventional battery cages) must be phased out by January 1st 2012. In
the meantime, cages have to provide 550 cm2 per hen since 2003. In newly built houses, hens
cannot be housed in conventional battery cages since 2003.
All member states had to implement Directive 1999/74/EC into their national
legislation before 1 January 2002. Belgium however only implemented it in October 2005
(Anon., 2005a). Because each country can be stricter than the minimum requirements
imposed by the Directive, there are a few exceptions on the time scheme. Sweden for instance
banned battery cages from 1999 onwards; Germany did so from the 1st of January 2010.
iii. Alternatives for conventional battery cages
From 2012 onwards, the housing of laying hens in the EU will be restricted to
enriched cages and to the so-called alternative systems. The main characteristics of these
systems are summarized below and in Table 1.
Table 1: Characteristics of enriched cages and alternative housing systems according EU Directive 1999/74/EC
Characteristic Enriched cages Alternative systems
Area per hen Min. 750 cm2 of which 600 cm
2 is usable area
1
Total cage area min. 2000 cm2
Max. 9 hens/m2
Height Usable area1: min. 45 cm
Other area: min. 20 cm
Max. 4 levels
Min. 45 cm between the levels
Floor Slope max. 14 % or 8° Must support the forward facing claws
Length feeders Min. 12 cm per hen Linear feeders: min. 10 cm/hen
Circular feeders: min. 4 cm/hen
Water supply Appropriate to group size
Min. 2 nipples or cups within the reach of each hen
Continuous drinking through: 2,5 cm/hen
Circular drinking through: 1 cm/hen
Claw-shortening devices Suitable devices No
Nest2 Yes Yes, min. 1 nest/7 hens
Group nest: min. 1 m2 for max. 120 hens
Litter3 Yes, such that pecking and scratching are possible Yes, min. 250 cm
2/hen
Min. 1/3 of the ground surface
Perches Yes, min. 15 cm/hen Yes, min. 15 cm/hen
1usable area: an area at least 30 cm wide with a floor slope not exceeding 14 %, with headroom of at least 45 cm. 2nest: a separate space for egg laying for an individual hen or for a group of hens 3litter: any friable material enabling the hens to satisfy their ethological needs
10 Chapter 1
Enriched cage
Enriched cages must provide 750 cm2 per hen, a nestbox, perches and litter such that
pecking and scratching are possible (Figure 3). Enriched cages exist in a wide variety of
group sizes, ranging from small (< 15 hens), over medium (15 – 30 hens) to large (> 30 hens)
(LayWel, 2006). Enriched cages are also
called modified or furnished cages. Although
the latter term is recommended because it is
thought to give the most accurate description
(Appleby, 2004; LayWel, 2006), throughout
this study the term „enriched‟ will be used in
accordance with the terminology of the
Directive.
The term „alternative‟ refers to a wide variety of non-cage housing systems. All these
systems are operated from inside and entered by the laying hen keepers. Because of the
possible confusion caused by the word „alternative‟ it has been stated that the term „non-cage‟
is more appropriate (Blokhuis et al., 2007). Therefore the term „non-cage‟ will be used
throughout this thesis. The following non-cage systems will be described: aviaries, floor-
raised systems, free-range systems and free-range organic systems.
Aviary system
Aviaries consist of the ground floor plus 1 to 4 levels of perforated platforms, from
which manure cannot fall on the hens below
(Figure 4). The headroom between the levels
must be at least 45 cm. The maximum
stocking density is 9 hens/m2 usable area.
Figure 3: Enriched or furnished cage
Figure 4: Aviary system
Chapter 1 11
Floor-raised system
Floor-raised or „barn‟ systems are single-level systems, meaning that there is only one
level for the birds at any point in the house. The maximum stocking density must not exceed 9
hens/m2 usable area. Each hen must have at
least 250 cm2 of littered area, the litter
occupying at least 1/3 of the ground surface.
Floor-raised systems may be combined with a
covered outdoor facility, the so-called
„Wintergarden‟ (Figure 5). This outdoor area is
connected to the hen house and has a concrete
floor, usually covered with litter.
Free-range system
Free-range systems are also single-level systems but they are always combined with
uncovered outdoor facilities (Figure 6). There must be several popholes giving direct access
to the outer area extending along the entire
length of the building. The outdoor run must
be of an area appropriate to the flock size and
it must be equipped with shelter from
inclement weather and predators.
Free-range organic
The organic laying hen husbandry is with regard to the design of the house similar to
free-range systems. However, there are some very important additional requirements. The
maximum stocking density must not exceed 6 hens/m2 usable area, instead of 9 in the other
systems. The maximum capacity of the hen house is restricted to 3.000 birds. Hens should
Figure 5: „Wintergarden‟
Figure 6: Free-range system
12 Chapter 1
have access to an outdoor run for at least 1/3 of their productive lifespan and each hen should
at least have 4 m2 of usable outdoor area.
In conclusion, it is clear that the above mentioned ban on conventional battery cages
aims to improve the welfare of laying hens (Wall et al., 2004). Although the influences of
these alternatives for conventional battery cages on laying hen welfare, productivity and user-
friendliness have been extensively evaluated and discussed (Abrahamsson and Tauson, 1995;
Tauson et al., 1999; Tauson, 2002; Rodenburg et al., 2005; 2008), it has also initiated the
question whether there aren‟t any adverse consequences of this decision on the spread and/or
persistence of infectious diseases in a flock. After all, one of the biggest advantages of
conventional battery cages is that, because hens are separated from their faeces, the risk for
disease transmission through faeces can be minimized (Duncan, 2000). For instance the
intensive and frequent hen-to-hen contacts and the presence of litter in non-cage systems
jeopardize the biosecurity and increase the risk for specific diseases to develop and spread.
For several infections such as E. coli, erysipelas and pasteurellosis an increase in incidence
has been observed after the move to non-cage systems (Hafez, 2001; Permin et al., 2002).
This leads to the question whether the same effect is to be expected for zoonotic pathogens
such as Salmonella.
Chapter 1 13
2. THE ROLE OF EGGS IN HUMAN SALMONELLOSIS
a. What is Salmonella?
In 1884, G. Gaffky was the first to culture Salmonella (Le Minor, 1994). Two years
later, D.E. Salmon gave his name to this bacterium when he succeeded, together with T.
Smith, in isolating Salmonella Choleraesuis from a pig (Salmon and Smith, 1886). Today, the
genus Salmonella encompasses a large taxonomic group with nearly 2500 recognized
serotypes or serovars (Heyndrickx et al., 2005). The most common way to classify isolated
Salmonella bacteria is serotyping according to the Kauffmann-White scheme, based on the
presence of H (flagellar), capsular (Vi) and O (somatic) antigens (Popoff and Le Minor, 1997).
Phage typing has been, and still is, a very important tool for the phenotypical characterization
of Salmonella strains, especially in outbreak investigations (Ward et al., 1987; Vieu, 2003;
Baggesen et al., 2010). Other ways of typing Salmonella strains are pulsed field gel
electrophoresis (Murase et al., 1995), random amplification of polymorphic DNA (Hilton et
al., 1996) and ribotyping (Grimont and Grimont, 1986).
From an epidemiological point of view, Salmonella serovars can also be divided into
groups based on their particular host specificity and pathogenesis (Kingsley and Bäumler,
2000). A first group consists of the host-restricted serovars. These serovars are capable of
causing severe systemic disease in a very limited number of related species. Typical examples
of these serotypes are S. enterica subsp. enterica serovar Gallinarum in poultry and S.
enterica subsp. enterica serovars Typhi and Paratyphi A in humans. A second group are the
so-called host-adapted serovars which are prevalent in one particular host species but which
can also cause disease in other host species (e.g. S. enterica subsp. enterica serovar Dublin,
causing severe disease in cattle but it may also cause disease in humans). Finally there is the
large group of the un-restricted or ubiquitous serovars (e.g. S. enterica subsp. enterica serovar
Enteritidis and S. enterica subsp. enterica serovar Typhimurium). These serovars only seldom
cause systemic infections in healthy adults but they are capable of colonising the gastro-
intestinal tract of a broad range of animals. The intestinal colonisation and often high levels of
faecal shedding in food producing animals allow these serovars to enter the food chain,
resulting from time to time in human cases of salmonellosis (Uzzau et al., 2000; Wallis and
Barrow, 2006).
Salmonella constitutes a genus of Gram-negative bacteria belonging to the family of
the Enterobacteriaceae, implying that strains belonging to this family are straight rods which
14 Chapter 1
generally are motile because of the presence of peritrichous flagella; they ferment glucose,
often with gas production; they reduce nitrate into nitrite and they are oxidase negative
(Grimont et al., 2000). Metabolic characteristics of the genus Salmonella are that urea is not
hydrolysed; tryptophan and phenylalanine are not deaminated; acetoin is not produced;
lactose, adonitol, sucrose, salicin and 2-ketogluconate are not fermented; H2S is produced
from thiosulphate; and lysine and ornithine are decarboxylated.
Until now, the focus of the European Salmonella control programs has been on S.
enterica subsp. enterica serovar Enteritidis (S. Enteritidis in short) and S. enterica subsp.
enterica serovar Typhimurium (S. Typhimurium), since these are the most commonly isolated
serovars in cases of human salmonellosis. In the next chapters, mainly Salmonella Enteritidis
will be discussed because of its typical relationship with eggs and egg products. Humans
acquire Salmonella Typhimurium via a much more diverse range of vectors such as pork
(Soumpasis and Butler, 2009), beef and poultry meat products (Dechet et al., 2006; Ethelberg
et al., 2007) and unpasteurized dairy products (Cody et al., 1999; Villar et al., 1999).
b. Infection pathway
i. Pathogenesis in chickens
The usual route of infection in chickens is the oral uptake of Salmonella bacteria from
the birds‟ environment. Contaminated feed and water for instance are important sources of
Salmonella infections in chickens (Cox et al., 1991; Heyndrickx et al., 2002). The vertical
transmission of Salmonella can also be an important issue in poultry: neonate chicks can get
infected by infected breeder hens and their eggs (Barrow, 1999; Poppe, 2000; Liljebjelke et al.,
2005). Once a few animals are infected, the infection can spread throughout the flock through
animal to animal contact (Byrd et al., 1998; Gast and Holt, 1999), through vectors such as
rodents and flies (Kinde et al., 2005; Carrique-Mas et al., 2009) or by airborne transmission
(Lever and Williams, 1996; Nakamura et al., 1997; Holt et al., 1998).
As in the pathogenesis of other bacteriological intestinal infections, the adhesion of the
Salmonella bacteria to the surface of the intestinal epithelial cells of the host is a very
important step in the interaction between the pathogen and the host (Finlay and Falkow, 1989).
This process occurs mainly through fimbriae, although some outer membrane proteins seem
to play a role as well (Fadl et al., 2002; Kingsley et al., 2002).
Chapter 1 15
Once attached, Salmonella stimulates its own uptake by the intestinal epithelial and
other cell types using a type III secretion system, encoded by the Salmonella Pathogenicity
Island I (SPI-1) (Darwin and Miller, 1999; Zhou and Galán, 2001). This is the invasion phase
of the pathogenesis. In chickens, this occurs mainly in the caeca (Desmidt et al., 1997; 1998).
Shortly after invasion, the intestinal epithelial cells will produce pro-inflammatory cytokines
(Kaiser, 1994; Klasing, 1998), attracting heterophilic granulocytes and macrophages (Van
Immerseel et al., 2002). The Salmonella bacteria will be phagocytised and are able to survive
and replicate in the macrophages. This also requires a type III secretion system regulated by
SPI-2 Through the spread of these infected macrophages, the bacteria will be able to reach
other internal organs such as the liver, spleen and the reproductive tract (Barrow and Lovell,
1991; Barrow, 1999). This is the systemic phase of infection.
ii. Clinical symptoms in chickens
Salmonella infections in chickens often cause only very limited symptoms or even no
symptoms at all. Two exceptions are Salmonella Gallinarum and Salmonella Pullorum,
serovars causing severe illness in chickens, with high morbidity and mortality in the flock
(Erbeck et al., 1993; Wong et al., 1996; Shivaprasad, 2003).
Young chicks can show symptoms of disease after infection with S. Enteritidis or S.
Typhimurium. The chicks may exhibit symptoms including anorexia, adipsia, general
depression, huddling together in small groups, immobility, ruffled feathers, white diarrhoea
and stained or pasted vents (Marthedal, 1977; McIlroy et al., 1989). Normally no typical
clinical symptoms occur in infected flocks of mature laying hens, although seldom a limited
increase in mortality may be observed (Humphrey et al., 1991; Kinde et al., 2000).
In general, the egg production in naturally infected laying hen flocks remains in the
normal range (Muller and Korber, 1992; Awadmasalmeh and Thiemann, 1993). However,
under experimental conditions a decreased egg production has been reported after oral (Gast
and Beard, 1992; Gast, 1994) or intravenous (Guard-Petter, 1998; Okamura et al., 2001)
infections of laying hens with S. Enteritidis.
iii. Mechanisms of egg contamination and numbers of infected eggs
As already mentioned above, consumption of contaminated eggs or egg products is the
main source of infection with S. Enteritidis for humans (Delmas et al., 2006; EFSA, 2006).
16 Chapter 1
The mechanisms of egg contamination have been extensively described by Gantois et al.
(2009). Below, only a short description of the main mechanisms of egg contamination will be
presented.
Generally, eggs can get contaminated by Salmonella bacteria via horizontal or vertical
transmission. Horizontal transmission means that the eggs get contaminated by penetration of
Salmonella through the egg shell during or after oviposition (Messens et al., 2005; De Reu et
al., 2006), with the bacteria coming from the colonized gut or from contaminated faeces. The
second possibility is vertical transmission, with direct contamination of the different egg
components before oviposition (Keller et al., 1995; Miyamoto et al., 1997; Okamura et al.,
2001). In this scenario the Salmonella bacteria originate from infection of the reproductive
organs.
For long, the percentage of eggs that are actually infected in an infected flock of laying
hens has been the topic of debate. Under experimental conditions, egg contamination rates
from Salmonella infected hens ranging from 0 % to 27.5 % have been reported, depending on
the inoculation route, the infection dose and the number of animals used (Keller et al., 1995;
Okamura et al., 2001). In naturally infected laying hen flocks, there is agreement that this
percentage varies strongly (Humphrey et al., 1989), although most studies indicate that it does
not exceed 3 % (Kinde et al., 1996; Schlosser et al., 1999).
Statistical techniques and quantitative risk assessment models are another way to
estimate the number of infected eggs. Going through the literature, estimates ranging from 1
out of 12.000 up to 1 out of 30.000 infected eggs can be found (Ebel and Schlosser, 2000;
Hope et al., 2002).
The number of contaminated eggs can also be determined at retail level. The Egg
Survey report of the Food Standards Agency (2004) studied the proportion of eggs
contaminated with Salmonella on the retail sale level by purchasing boxes of 6 eggs from a
cross-section of retail outlets throughout the UK. It showed that in 0.34 % (95 % CI 0.17 –
0.62 %) of the 4.753 boxes tested at least one egg out of the 6 was positive for Salmonella. A
smaller study with similar aims did not detect any Salmonella in the contents of 100 dozens
eggs (Schutze et al., 1996).
The number of infected eggs in a flock is logically related with the number of infected
hens in that flock and the timing of egg production relative to infection: eggs produced soon
after infection are much more likely to harbour Salmonella (Gast and Beard, 1990). However,
Chapter 1 17
it is very hard to gain accurate information on the within-flock prevalence of Salmonella in
laying hen flocks. This lack of knowledge complicates the quantitative assessment of several
management and preventive measures at the level of the primary production.
c. Human Salmonella infections
i. Evolution in time
Already for many decades salmonellosis is recognised worldwide as an important
foodborne disease, even becoming more common because of the globalization of the food
supply and changes in lifestyle (Todd, 1997). The most recent data on zoonoses of the
European Food Safety Authority indicate that Salmonella is the second most important
foodborne disease in the EU, only preceded by Campylobacter (EFSA, 2010). In 2008, there
were 131.468 confirmed cases of human salmonellosis in the 27 EU member states. In the
European Community, the number of human salmonellosis cases shows a statistically
significant decreasing trend in the past 5 years (Figure 7). However, this is only the case in 10
out of the 27 member states. In several countries no significant difference could be observed
during this period, whereas in 7 member states even a significant increase was observed.
In 2008, S. Enteritidis was the most commonly reported serovar in the EU (58.0 % of
all confirmed cases), followed by Salmonella Typhimurium (21.9 %) (EFSA, 2010). It is
worth mentioning that the number of S. Enteritidis cases tends to decrease on the EU level,
increasing the relative proportion of S. Typhimurium cases. Specific for Belgium, a
0
50000
100000
150000
200000
250000
2004 2005 2006 2007 2008
Nu
mb
er o
f co
nfi
rmed
cas
es o
f sa
lmo
nel
losi
s
Year
Figure 7: Evolution in number of confirmed cases of human salmonellosis in
the EU (EFSA, 2010)
18 Chapter 1
downward trend in numbers of human salmonellosis cases can be observed, with 3.944
confirmed cases in 2008 (National Reference Centre for Salmonella and Shigella, 2009).
Since the peak of 15.774 cases in 1999, a decrease in number of human salmonellosis cases in
Belgium has been observed with a considerable drop from 2005 onwards, strongly associated
with a drastic decrease of S. Enteritidis cases (Collard et al., 2008). It is thought that the
vaccination of commercial laying hens against Salmonella has played a huge role in this
evolution. In Belgium, S. Typhimurium has become the most commonly isolated serotype,
accounting for 57.78 % of the cases. S. Enteritidis came second with 20.89 % (National
Reference Centre for Salmonella and Shigella, 2009).
ii. Relationship between eggs and human infections
Although humans can get infected by Salmonella through a wide range of food
products, both from animal and non-animal origin, eggs and egg products are generally
considered as the major sources of infection with S. Enteritidis, the most common cause of
human salmonellosis worldwide (Rodriguez et al., 1990; Angulo and Swerdlow, 1999; Poppe,
2000; Patrick et al., 2004; Namata et al., 2008).
An illustration of the strong relationship between eggs and human Salmonella
(Enteritidis) infections is the observation that the increase in number of S. Enteritidis isolates
in humans is directly related with an increase in number of isolates from eggs and chicken
meat (Lee, 1974; Rabsch, 2000). The studies of McIlroy and Mc Craken (1990) and Van
Duijkeren et al. (2002) both showed a drastic increase in S. Enteritidis isolates from chickens
in the United Kingdom and The Netherlands respectively during the 1980‟s, coinciding with
the worldwide rise of S. Enteritidis outbreaks in humans (Hogue et al., 1997). The opposite is
also true: reducing Salmonella flock prevalence results in a directly proportional reduction in
human health risk (Altekruse et al., 2003).
iii. Clinical symptoms and economic relevance of salmonellosis in
humans
The incubation period of a Salmonella infection in humans varies from 5 hours to 7
days (Plym Forshell and Wierup, 2006), but clinical signs usually begin 12 to 36 hours after
consumption of contaminated food (Omwandho and Kubota, 2010). Diarrhoea, abdominal
pains, vomiting, headache and fever are the most common symptoms (Steinert et al., 1990;
Anon., 1992). The duration of illness varies from 4 to 10 days but faecal shedding can last 4
Chapter 1 19
to 7 weeks in adults and children respectively (Buchwald and Blaser, 1984). Treatment with
antimicrobials does not seem necessary in cases without complications because of the risk of
prolonging the carrier state (Dixon, 1965; Aserkoff and Bennett, 1969). Especially in the
population of the so-called YOPI‟s (i.e. the young, the older ones, the pregnant and the
immune deficient), septicaemia may occur leading to meningitis, arthritis, osteomyelitis,
pneumonia and pericarditis. Occasionally a Salmonella infection might even result in death
(Hohmann, 2001; McCabesellers and Beattie, 2004). Subclinical infections and/or carriers
also occur and investigations found that 7 % to 66 % of infected humans are subclinical
carriers (Plym Forshell and Wierup, 2006; and references therein).
Looking at the age distribution (Figure 8), it is clear that the highest number of
confirmed cases per 100.000 population can be found in the group of the 0 to 4 years old. This
is 3 times higher than in the group of the 5 to 14 year old and 6 to 9 times higher than in the
other age categories. A slight increase can be seen in the group of 65 years and older (EFSA,
2010).
Human Salmonella infections also have a significant economical impact (e.g. direct
medical and non-medical costs, temporary absence from school or work...). A study in The
Netherlands estimated the costs for Salmonella infections during 1999 at 4 million € (van den
Brandhof, 2004). Roberts et al. (2003) concluded that hospital costs were the highest for
Salmonella compared to other infectious intestinal diseases. An American study (Bryan and
Doyle, 1995) estimated that the costs per case of human salmonellosis ranges from
0
20
40
60
80
100
120
0-4 5-14 15-24 25-44 45-64 ≥65
Co
nfi
rmed
cas
es p
er 1
00,0
00
po
pu
lati
on
Age (in years)
Figure 8: Age-specific distribution of confirmed cases of human salmonellosis
(EFSA; 2010)
20 Chapter 1
approximately US $ 40 for uncomplicated cases to US $ 4.6 million for those ending with
hospitalization and death.
In this context, it should be mentioned that presumably the true prevalence of human
Salmonella infections is much higher, as many cases of salmonellosis are not reported
because the ill person either does not visit a physician, no sample is obtained for laboratory
tests or the laboratory findings are not registered in national data bases (Rabsch et al., 2001).
Voetsch et al. (2004) estimated that for each laboratory-confirmed case of Salmonella, there
are 38 cases that are not ascertained through surveillance.
Chapter 1 21
3. EPIDEMIOLOGY OF SALMONELLA
a. Prevalence of Salmonella in laying hens: the EFSA baseline study
In order to provide a scientific basis for setting Salmonella reduction targets on
commercial large-scale laying hen farms a good indication of the prevalence was needed.
Therefore the European Commission ordered an EU-wide Salmonella baseline study
(Commission Decision 2004/665/EC) (Anon., 2004a) co-ordinated by DG SANCO and the
European Food Safety Authority (EFSA). In each member state the survey, carried out by the
official authorities, implied that a random group of commercial laying hen farms, stratified by
the total capacity of the farms, was sampled. Only farms with a capacity of more than 1000
laying hens were sampled. From each farm only one randomly selected flock was sampled
within nine weeks of depopulation. From this flock, 5 pooled faeces samples and 2 mixed dust
samples were collected. A flock was considered positive if in one or more of the seven
samples Salmonella could be detected. All specifications of this survey were prescribed by the
technical document of the European Commission DG SANCO (Anon., 2004b).
The objectives of this baseline study were five-fold: a) to estimate the prevalence of
Salmonella in commercial laying hen flocks, both at the EU level and the level of the
individual members states b) to estimate the prevalence of S. Enteritidis and S. Typhimurium
at farm level c) to investigate the serovar distribution, both at the EU level and the level of the
individual members states d) to determine potential risk factors for the presence of Salmonella
on laying hen farms and e) to evaluate the precision and the accuracy of the sampling design
with regards to the prevalence estimates (EFSA, 2007).
The final dataset contained data from 23 EU member states and Norway, 5.310 laying
hen farms in total. For several reasons, no data from Malta and Slovakia were included. All
samplings took place between October 2004 and September 2005.
Salmonella could be detected on 30.8 % of the laying hen farms in the EU, although large
differences between individual member states could be observed with prevalences ranging
from 0 % to 79.5 %. Specifically looking at S. Enteritidis and/or S. Typhimurium, 20.4 % of
the laying hen farms were positive at the EU level, ranging from 0 % to 62.5 % (EFSA, 2007).
The observed prevalences of all participating countries are presented in Table 2. The three
most commonly found serovars were S. Enteritidis, S. Infantis and S. Typhimurium, with S.
Enteritidis counting for 60 % of the isolates on the positive farms.
22 Chapter 1
The housing of laying hens in battery cages turned out to be a risk factor. However,
confounding with other variables such as the flock size could not be ruled out. Vaccination
against Salmonella on the other hand turned out to be a protective factor, although this
beneficial effect could mainly be observed in countries with a high Salmonella prevalence
(EFSA, 2007).
In the framework of the baseline study, 141 Belgian farms were sampled, resulting in a
Salmonella prevalence of 37.6 %. S. Enteritidis and/or S. Typhimurium were found on 27.7 %
of the farms.
Table 2: Prevalence of Salmonella-positive laying hen farms in the EU (EFSA, 2007)
Salmonella spp. Salmonella Enteritidis and/or Typhimurium
Country No. of flocks % pos. CI 95% % pos. CI 95%
Austria 337 15.4 12.7-18.5 10.7 8.4-13.4
Belgium 141 37.6 31.4-44.1 27.7 22.1-33.9
Cyprus 25 28.0 21.7-33.0 8.0 3.7-12.3
Czech Republic 64 65.6 61.3-68.2 62.5 58.0-65.2
Denmark 185 2.7 1.6-4.3 1.6 0.8-3.0
Estonia 11 18.2 -* 9.1 -
Finland 250 0.4 0.0-1.6 0.4 0.0-1.6
France 511 17.2 14.6-20.2 8.0 6.2-10.3
Germany 553 28.9 25.7-32.3 24.2 21.2-27.5
Greece 140 49.3 42.8-55.5 25.7 20.5-31.6
Hungary 267 43.8 39.9-47.6 33.7 30.0-37.4
Ireland 146 1.4 0.6-2.6 0.0 0.0-0.7
Italy 367 29.2 25.4-33.1 7.9 5.9-10.5
Latvia 6 16.7 1.0-46.8 0.0 0.0-29.1
Lithuania 9 44.4 22.6-62.9 44.4 22.6-62.9
Luxembourg 9 0.0 - 0.0 -
Poland 328 76.2 72.0-79.9 55.5 50.8-60.0
Portugal 44 79.5 66.7-87.7 47.7 34.9-60.4
Slovenia 98 19.4 15.4-23.8 9.2 6.4-12.7
Spain 485 73.2 70.1-76.0 51.5 48.2-54.8
Sweden 168 0.0 0.0-1.3 0.0 0.0-1.3
The Netherlands 409 15.4 12.6-18.6 7.8 5.9-10.4
United Kingdom 454 11.9 9.9-14.7 7.9 6.2-10.1
EU 5007 30.8 29.8-31.8 20.4 19.5-21.3
Norway 303 0.0 0.0-0.8 0.0 0.0-0.8
*No confidence interval for Estonia and Luxembourg since all farms in these member states were sampled
24 Chapter 1
b. Prevalence of Salmonella in laying hens: the Belgian situation
i. Breeding parents flocks
Since Regulation No. 1003/2005 (Anon., 2005b) became valid, implying that the
prevalence of five Salmonella serovars of public health importance (i.e. S. Enteritidis, S.
Typhimurium, S. Hadar, S. Infantis and S. Virchow) in breeding flocks of more than 250 hens
may not exceed 1 %, the prevalence of the targeted serovars in Belgian breeding flocks
decreased from 4.2 % in 2004 to 0.9 % in 2008, which is also the average at the EU level
(EFSA, 2007; EFSA, 2010). It is however remarkable that 7.3 % of the breeding flocks were
positive for serovars other than the five targeted ones, which is above the 1.8 % at the EU
level (EFSA, 2010).
ii. Laying hen flocks
In laying hen flocks, the prevalence of S. Enteritidis and S. Typhimurium decreased
dramatically from 27.2 % in 2004 to 3.7 % in 2008, which is below the mean prevalence of
5.9 % at the EU level (EFSA, 2007; EFSA, 2010). The drop in Salmonella prevalence was
most pronounced in 2005, an observation which has been attributed to the vaccination of the
laying hens: although the vaccination of laying hens against Salmonella only became
mandatory in 2007, the Belgian Federal Agency for the Safety of the Food Chain already
strongly recommended vaccination since early 2005, and many companies implemented this
measure (Collard et al., 2008). As is the case in Belgian breeding flocks, also during the
rearing and the production period more flocks are found positive for serovars other than S.
Enteritidis and S. Typhimurium than the EU average (8.0 % vs. 2.3 %) (EFSA, 2010).
c. Risk factors for the presence of Salmonella in laying hen flocks
In addition to the detection of Salmonella in laying hen flocks and the monitoring in
time, also the prevention of Salmonella contamination of laying hens is of the utmost
importance. This requires knowledge on the risk factors associated with its presence on laying
hen farms. Several risk factors for the prevalence of Salmonella in laying hens have been
identified in observational and experimental studies.
Chapter 1 25
The main risk factors are:
The system in which the hens are housed has often been described as a risk factor
but the published studies show contradictory results, ranging from a preventive
effect of the conventional battery cage system (Mollenhorst et al., 2005) over no
influence (Schaar et al., 1997) up to a higher risk of Salmonella in battery cages in
comparison to non cage system (Mølbak and Neimann, 2002; EFSA, 2007). When
interpreting these results, three things have to be taken into account. First, there
were large differences in sample size and methodology used between all studies.
Secondly, none of the mentioned studies was specifically set up to determine the
influence of housing systems on the Salmonella prevalence in laying hen flocks.
Finally, factors such as farm and flock size and vaccination status of the flock
presumably play a confounding role. All this makes it very hard to draw a
consistent conclusion on the influence of the housing system on the prevalence of
Salmonella in laying hen flocks.
Stress is shown to have an immunosuppressive effect in laying hens (El-Lethey et
al., 2003; Humphrey, 2006), which can have negative consequences with respect
to Salmonella infection and shedding. There are several moments in the laying
hen‟s life where the bird is subjected to stress: moving from the rearing site to the
egg producing plant (Hughes et al., 1989), the onset of lay (Jones and Ambali,
1987; Humphrey, 2006), final stages of the production period, thermal extremes
(Thaxton et al., 1974; Marshally et al., 2004) or transportation to the
slaughterhouse (Beuving and Vonder, 1978). The stress associated with induced
moulting potentially has a harmful influence on the Salmonella status of the flock
(Holt, 2003).
The persistence of Salmonella on a farm is also a significant risk factor, even to
the extent that it is thought that the major part of Salmonella-infections on laying
hen farms are not newly introduced on the farm but are the result of re-
introduction of the pathogen from the farm‟s environment (van de Giessen et al.,
1994; Gradel et al., 2004; Carrique-Mas et al., 2009b). This observation underlines
the importance of an adequate cleaning and disinfection policy. Nevertheless,
because of their intrinsically complicated structures, laying hen houses are
notoriously difficult to clean and disinfect (Wales et al., 2006).
26 Chapter 1
The role of rodents, flies, beetles and wild birds as vectors in the transfer of
Salmonella has been extensively discussed (Guard-Petter, 2001; Davies and
Breslin, 2001; 2003; Kinde et al., 2005; Stenzel et al., 2008; Carrique-Mas et al.,
2009a). Another very important pest in laying hens‟ houses is the poultry red mite
(Dermanyssus gallinae). It has been shown under experimental conditions that
mites could play a role in the persistence of Salmonella in laying hens, either by
transferring the bacterium from hen to hen or by hens consuming contaminated
mites leading to a persisting infection (Valiente-Moro et al., 2007; Valiente-Moro
et al., 2009). Whether this is also the case under field conditions and in non-battery
cage housing systems still remains unknown.
Finally, the use of vaccination against Salmonella has beyond doubt a significant
protective influence on shedding of Salmonella in laying hen flocks since the
currently available vaccines reduce both the shedding and colonization of the
reproductive tract, leading to a decrease in the number of internally contaminated
eggs (Feberwee et al., 2001; Woodward et al., 2002; Gantois et al., 2006).
d. Salmonella control programs in laying hen flocks
i. Legislative background
The dramatic increase in human S. Enteritidis cases worldwide since the 1980‟s has
been associated with the rise of S. Enteritidis in poultry between the 1960‟s and the 1980‟s
(Hogue et al., 1997; Rabsch et al., 2000). Because of this important role of poultry in the
epidemiology of human salmonellosis, the European Parliament issued Council Directive
92/117/EEC (Anon., 1992). The requirements of this directive were three-fold. Member states
should a) monitor for zoonotic agents b) take steps to reduce the risk of introducing
Salmonella on the farm and c) control Salmonella in flocks of parents breeding hens.
Although this directive was a fair attempt to tackle the swift rise of human salmonellosis in
the EU, the benefit to human health was rather limited. One of the reasons for this is that the
main focus was on the control of the vertical transmission of Salmonella, with very strict
measures at the level of the grand parent and parent breeding flocks. However, both for
invasive and non-invasive serotypes the horizontal transmission between animals is at least as
important as the vertical transmission. Therefore the top-down approach pushed forward in
this directive was by far insufficient to control Salmonella on the level of the laying hen farms.
Chapter 1 27
In order to fine-tune the measures of this first directive, the EU parliament adopted
Regulation No. 2160/2003 which stated that proper and effective measures must be taken in
the member states to detect and control Salmonella at all relevant stages of production,
processing and distribution, particularly at the level of primary production (Anon., 2003a).
Each type of measure at each stage has some importance in the reduction of Salmonella, but
no measure is successful on its own. A subsequent directive (2003/99/EC) stated that zoonotic
agents and their antimicrobial resistance must be strictly monitored (Anon., 2003b). The
emphasis of the European approach in controlling Salmonella contamination of eggs is on the
prevention and monitoring during the live-phase, i.e. the rearing of the laying hens and the
egg production at the laying hen farms.
One of the spearheads of the above mentioned legislation is that EU member states
need to establish national Salmonella control programs (NCP‟s). Minimal requirements for
NCP‟s in commercial laying hen flocks were laid down in Regulation No. 1091/2005 (Anon.,
2005c). They comprise that a) antimicrobials cannot be used to control Salmonella b) in
member states where the prevalence of S. Enteritidis in commercial laying hens is higher than
10 %, vaccination against S. Enteritidis is mandatory and c) live vaccines can only be used
during the rearing period of the pullets and the manufacturer has to provide a method to
distinguish the vaccine from field strains of S. Enteritidis.
The surveillance data, available since the application of Directive 92/117/EEC, made it
possible to set reduction targets for the prevalence of Salmonella in parents breeding flocks.
These reduction targets were laid down in Regulation No. 1003/2005 (Anon., 2005b),
implying that the prevalence of 5 Salmonella serovars of public health importance in breeding
flocks of more than 250 hens may not exceed 1 %. One of the measures to achieve this is the
mandatory sampling of all breeder flocks and hatcheries since 1 January 2007. If S. Enteritidis
or S. Typhimurium is detected, the eggs from these flocks can no longer be hatched and the
hens are culled or subjected to sanitary slaughter. If S. Infantis, S. Virchow or S. Hadar is
detected, the farmer has to draw up a specific action plan in order to eliminate infection and
prevent dissemination. Non-members states of the EU supplying hatching eggs or live poultry
for breeding to the EU must have submitted a Salmonella control program which is
considered equivalent to the EU provisions.
In a next step, annual reduction targets for S. Enteritidis and S. Typhimurium in all
commercial laying hen flocks (both pullets and egg producing chickens) were set by
Regulation No. 1168/2006 (Anon., 2006). The reduction targets differ from member state to
28 Chapter 1
state, depending on the result of each country in the EFSA baseline study from 2004 - 2005.
For the countries with a S. Enteritidis and S. Typhimurium combined prevalence lower than
10 %, an annual reduction of 10 % was required. When the combined prevalence was 10-
19 %, 20-39 % and higher than 39 % the annual reduction targets were set at 20 %, 30 % and
40 % respectively.
This regulation also gives detailed information on the sampling programs for laying hen
flocks. Rearing flocks should be sampled a) at day old (which means on arrival of the chicks
from the hatchery) and b) 2 weeks before entering the egg laying phase using 2 pairs of boot
swabs in non-cage rearing systems per house or 60 faeces samples of 1 g in cage reared
pullets. Laying hen flocks must be sampled every 15 weeks during the entire production cycle
from the age of 22 - 26 weeks onwards. From laying hens in cage systems, 2 pooled faeces
samples must be collected, flocks in non-cage housing systems have to be sampled using 2
pairs of boot swabs. All obtained samples have to be analyzed in an officially approved
laboratory, following the ISO 6579:2002 (Annex D) method. In addition to this, farms with a
capacity of >1000 hens have to be sampled by the official authorities meaning that from each
farm yearly 1 randomly selected flock has to be tested using 2 pooled faeces samples or 2
pairs of boot swabs and 1 mixed dust sample. A flock is regarded as infected with Salmonella
if at least 1 sample from the laying hens or 1 relevant environmental sample tests positive.
Furthermore, Regulation No. 1237/2007 laid down restrictions concerning the trade of
table eggs from flocks which are infected with S. Enteritidis or S. Typhimurium, saying that
from February 2009 onwards, eggs from any flock infected with these 2 serovars will be
banned from the market, unless they are treated in a manner that guarantees that all
Salmonella bacteria are destroyed. Because of the considerable economical implications, the
regulation gives the farmer the opportunity to dispute positive sampling results. This can be
achieved by the testing, at the producer‟s expense, of either a) seven faecal/environmental
samples b) 4.000 eggs from the affected flock or c) 300 hens tested for the presence of
Salmonella in their caeca and ovaries. When the results of these additional tests are negative,
the original findings become invalid.
ii. Detecting Salmonella in laying hen flocks: the chicken or the egg?
The detection of Salmonella in laying hen flocks is one of the cornerstones of the
national Salmonella control programs imposed by the EU. Detecting and monitoring the
presence of Salmonella allows taking necessary and sometimes corrective steps (e.g. adequate
Chapter 1 29
cleaning and disinfection, rodent control, vaccination...) to combat Salmonella and at the same
time it generates useful information on the prevalence of Salmonella and its evolution in time
on the population level.
However, the efficiency of such control programs is highly dependent on the accuracy
of the chosen sampling method (Fletcher, 2006) and the sensitivity of the culture methods
(Carrique-Mas and Davies, 2008). The total number, volume and frequency of the collected
samples all influence the sensitivity of a sampling protocol. Especially in the case of a low
within-flock prevalence higher numbers of samples are necessary but there are obviously
several logistic and economical issues that need to be taken into account when setting up a
control program. One of the major merits of the EU in its crusade against Salmonella is the
harmonisation and coordination of the different monitoring and control programs that were
installed in the different members states by drawing up Regulation No. 1168/2006, stating that
laying hen flocks must be sampled throughout the production cycle using 2 pooled faeces
samples (in cage flocks) or 2 pairs of boot swabs (in non-cage systems).
Besides the sampling protocol imposed by Regulation No. 1168/2006, a wide variety
of different methodologies for the sampling of Salmonella have been used in different
countries and different time periods. Summarized, the investigation of Salmonella in poultry
at the primary production stage may involve either the collection of samples from the laying
hens themselves such as eggs, blood, cloacal swabs and caecal samples or the collection of
material from the environment in the poultry house such as faeces, dust and litter (Carrique-
Mas and Davies, 2008a).
Since eggs are the main vectors for human Salmonella (Enteritidis) infections, it seems
plausible to focus the sampling of laying hen flocks on the bacteriological detection of
Salmonella in eggs. However there are some major drawbacks making the testing of eggs very
labour-extensive and expensive (Carrique-Mas and Davies, 2008b). Firstly, there is a very low
rate of egg contamination, even in flocks known to be infected with S. Enteritidis. Secondly,
the number of Salmonella bacteria deposited in contaminated eggs is believed to be low,
making the bacteriological detection challenging. Finally, the fact that in many countries
commercial laying hens are vaccinated against Salmonella further hampers the bacteriological
testing of eggs as a useful methodology because of the strongly reduced shedding of the
pathogen by vaccinated hens.
30 Chapter 1
The use of caecal and ovarial samples from hens post-mortem is regarded as a very
accurate method to detect Salmonella in laying hen flocks (Nief and Hoop, 2008) although
practical and economical factors impose limitations on the user friendliness (Barnhart et al.,
1993). According to Regulation No. 1237/2007, the caeca and/or ovaries of 300 hens in a
flock can be analyzed when a positive test result from the standard control program is
disputed by the farmer.
Serology has been used in Salmonella control programs, for instance in Denmark
(Wegener et al., 2003). Combined with bacteriology it increases the sensitivity of the testing
protocol. However, serology is no longer a usable tool in many countries because of the
vaccination of laying hen flocks against Salmonella and the cross-reactivity between vaccine
induced and natural antibodies.
Cloacal swabs have the advantage that they directly originate from the laying hen,
giving information on the current Salmonella status of the flock. However, the low sensitivity
is a very important disadvantage because of the intermittent shedding of the bacteria by
infected hens (Van Immerseel et al., 2004) and the very limited amount of faecal material
tested (Bichler et al, 1996). To circumvent this, a large number of cloacal swabs should be
taken in a flock but this implies practical problems and causes agitation among the birds.
One of the most commonly used methods is the testing of fresh faeces. This can be
done by collecting naturally pooled fresh faecal material or by collecting and pooling
individual piles of fresh faeces. Wales et al. (2006) stated that the inclusion of larger volumes
of mixed faecal material from a larger number of hens enhances the detection of Salmonella.
On the other hand, many authors also suggested that there might be a dilution effect when
positive and negative faecal material is mixed for culture (Kivelä et al., 2007; Arnold et al.,
2010; Singer et al., 2009).
The intensive sampling of the laying hen‟s environment is also regarded as an
effective method to detect Salmonella in laying hen flocks (Aho, 1992; Musgrove et al., 2005;
Carrique-Mas et al., 2009). From time to time the relevance of environmental sampling has
been disputed because the detection of Salmonella in a sample from the poultry house‟s
internal environment may originate from other sources such as wildlife and feed, rather than
reflecting the actual infection status of the flock. Despite these remarks there is general
agreement on the value of environmental sampling and the chance to gain interesting
Chapter 1 31
information on the infection status of the flock can even be increased by including vectors
such as rodents in the sampling (Kinde et al., 2005; Wales et al., 2007). A wide range of
environmental samples have been described such as dust (Davies and Wray, 1996; Davies and
Breslin, 2003), litter (Kingston, 1981), boot/sock swabs (Skov et al., 1999; McCrea, 2005),
drag swabs (White et al., 1997; Castellan et al., 2004) and hand-held gauze swabs (Davies and
Wray, 1996; Zewde et al., 2009).
The efficiency of a certain sampling method is also dependant on other factors such as
the vaccination status of the flock and the stage of lay. It has been thought that there is more
chance to detect Salmonella in a flock as the flocks becomes older, although little is known
about the underlying mechanisms (Garber et al., 2003; EFSA, 2007). Vaccines claim to
reduce the shedding of Salmonella, but this possibly also reduces the detection of infected
flocks because of the lower number of shed organisms and the lower within-flock prevalence
in vaccinated flocks (Van Immerseel et al., 2004). This raises the question whether the
currently used sampling methodologies in the Salmonella monitoring programs of several EU
member states are accurate enough to detect Salmonella in low prevalence flocks.
32 Chapter 1
4. ANTIMICROBIAL RESISTANCE IN LAYING HENS
Another „hot topic‟ is the origin and spread of antimicrobial resistance, both in animals
and humans. In this context, the transfer of antimicrobial resistance from food producing
animals to humans is, along with the microbiological integrity of the food, gaining weight as
an important aspect of food safety.
More and more concerns have raised on the systematic use of antimicrobials in animal
food production. The emergence of antimicrobial resistance initiated by this use compromises
animal health and welfare because of therapy failure. In addition to this, there are also some
possible adverse consequences for public health because of the spread of antimicrobial
resistance from animals to humans, leading to treatment failure in human infectious diseases
(EFSA, 2009; Jordan et al., 2009).
There are several pathways by which the presence of antimicrobial resistance in food
derived from animals could have potential human health implications a) antimicrobial
resistant zoonotic pathogens (e.g. Salmonella) in contaminated food cause a human infection
that requires antibiotic treatment and therapy is compromised b) antimicrobial-resistant
bacteria non-pathogenic to humans are selected in the animals and when contaminated food is
ingested, the bacteria transfer resistance determinants to other commensal and potential
pathogenic bacteria in the human gut and c) antibiotics remain as residues in food products,
allowing the selection of antibiotic-resistant bacteria after the food is consumed (Piddock,
1996). Another route via which resistant bacteria can be transmitted is through direct contact.
Similar resistance patterns have been described in animals and the farmers and slaughterhouse
workers handling and processing them, indicating circulation of bacterial genetic material
between the animals and humans (Ozanne et al., 1987; Nijsten et al., 1994; van den Bogaard
et al., 2001; 2002). From this point of view, the transfer of resistance from animals to humans
can be seen as an occupational disease (Cole et al., 2000).
To fully assess the size of the problem of antimicrobial resistance in food animal
husbandry, the EU adopted Directive 2003/99/EC (Anon., 2003b), implying that member
states must monitor and report on antimicrobial resistance in Salmonella and Campylobacter
isolates from animals and food. However, there are still some major drawbacks. A first
weakness is that in many of these programs the monitoring of antimicrobial resistance is
restricted to pigs, cattle and broilers (Aarestrup, 2004). Laying hens are either not sampled, or
Chapter 1 33
the data from hens are merged with the results of the broilers. This can be explained by the
fact that in laying hens the use of antimicrobials is relatively sparse, especially during their
productive life on the egg-producing plant, giving the impression that laying hens are of
negligible importance within the whole issue. Nonetheless, the consumption of eggs and -to a
lesser extent- meat of culled hens can pose a public health risk. Secondly, the obligation to
monitor and report only applies for zoonotic pathogens, the monitoring of resistance data
from indicator bacteria is voluntary.
All this explains why the published data for laying hens primarily report about
antimicrobial resistance in zoonotic pathogens such as Salmonella and Campylobacter spp. or
from bacteria with a clinical relevance for laying hens such as Avian Pathogenic E. coli and
Pasteurella multocida (Gyles, 2008; Huang et al., 2009).
When comparing results of the monitoring of resistance in zoonotic and pathogenic
bacteria, some possible sources of bias are present such as varying selection criteria used over
time and varying participation among veterinarians. Moreover, some infections are more
likely to generate isolates than others and isolates from some infections are more likely to be
sent for susceptibility testing. In many cases, treatment will already have been started when
isolates are collected and sent to the laboratory. Furthermore, inclusion of only clinical
isolates could lead to an overestimation of the occurrence of resistance because veterinarians
will often only send samples after they have experienced treatment failure (Aarestrup, 2004;
Bywater, 2005; Wallmann, 2006). Therefore, the monitoring of resistance in indicator bacteria
is of utmost importance in surveillance programs.
Indicator bacteria are generally regarded as useful tools to monitor antimicrobial
resistance since they can be isolated from healthy animals, giving a hint of the level of
resistance in a particular population and they are a potential source of resistance genes that
can spread horizontally to zoonotic and other bacteria through the food chain (Winokur et al.,
2001; Wang et al., 2006). In addition, they acquire antimicrobial resistance faster than other
commonly found bacteria, implying that changes in the resistance of these species serve as a
good indicator of resistance in potentially pathogenic bacteria (Aarestrup, 2004; WHO, 2007;
Miranda et al., 2008). Commensal Escherichia coli and Enterococcus faecalis are
internationally used as respective Gram-negative and Gram-positive indicator bacteria
because of their common presence in the animal and human intestinal tract (Wray and
Gnanou, 2000; Sørum and Sunde, 2001; de Jong et al., 2009). Besides this, both Escherichia
34 Chapter 1
coli and Enterococcus faecalis are associated with disease in humans. For E. coli, urinary tract
infection, sepsis/meningitis and enteric/diarrheal disease are the 3 general clinical syndromes
associated with human infection (Nataro and Kaper, 1998; Stenutz et al., 2006). E. faecalis
causes 80 % to 90 % of human enterococcal infections (Teixeira and Facklam, 2003). The
most commonly observed human infections by enterococci are urinary tract infections, pelvic
and intra-abdominal wound infections, bacteriaemia and endocarditis.
Till now, the number of studies describing antimicrobial resistance in indicator
bacteria from healthy laying hens or shell eggs is very limited. Moreover, differences in size
and methodology make it difficult to gain a clear insight into the situation in laying hens. An
overview of the studies describing antimicrobial resistance in healthy laying hens is presented
in Table 4.
The move to housing systems different from the conventional battery cages, as imposed
by the EU, can potentially influence antimicrobial resistance patterns. Both for poultry and
other animal species it has been suggested that the move from conventional indoor production
towards free-range and organic production exerted a beneficial effect on the levels of
antimicrobial resistance in zoonotic and indicator bacteria (Avrain et al., 2003; Thakur and
Gebreyes, 2005; Ray et al., 2006, Schwaiger et al., 2008; 2010, Young et al., 2009). On the
other hand it has been shown that the move to non-cage housing systems for laying hens
resulted in an increased incidence of particularly bacterial diseases (Fossum et al., 2009;
Kaufmann-Bart and Hoop, 2009), which could potentially lead to increased antibiotic usage.
It is therefore necessary to investigate the impact of these new laying hen housing systems on
the prevalence of antimicrobial resistance and to monitor antimicrobial resistance
development in laying hens.
Table 4: Overview of the studies describing antimicrobial resistance in healthy laying hens
Reference Study
country
Bacterium Sample type No. of isolates
(no. of farms)
Methodology
of testing
Cut-off
Yoshimura et al. (2000)
Japan
E. faecalis and E. faecium
Faecal droppings
222 (34)
MIC
JSC
Musgrove et al. (2006) USA E. coli Shell eggs 194 (N.A.) MIC CLSI
Kojima et al. (2009) Japan E. coli
E. faecalis
E. faecium
Fresh faecal samples
Fresh faecal samples
Fresh faecal samples
530 (N.A.)
251 (N.A.)
159 (N.A.)
MIC
MIC
MIC
JSC and CLSI
JSC and CLSI
JSC and CLSI
Schwaiger et al. (2008) Germany E. coli Cloacal swabs 533 (20) MIC DIN
Schwaiger et al. (2010) Germany E. faecium
E. raffinosus
E. faecalis
Cloacal swabs
Cloacal swabs
Cloacal swabs
84 (20)
439 (20)
328 (20)
MIC
MIC
MIC
DIN
DIN
DIN
CLSI: Clinical laboratory Standards Agency
DIN: Deutsches Institut für Normung
JSC: Japanese Society for Chemotherapy N.A.: not available
36 Chapter 1
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CHAPTER 2
SCIENTIFIC AIMS
Chapter 2 57
Salmonella is one of the most common bacterial causes of human disease in the
European Union. Eggs and egg products remain one of the major sources of infection for
humans.
The EU decision to ban conventional battery cages was based on the concerns about
laying hen welfare. Whether this move to alternative non-cage systems will also influence the
prevalence and persistence of zoonotic pathogens, such as Salmonella, in the flock has not yet
been thoroughly examined.
For several animal species such as pigs and cattle it has been described that the
farming type (e.g. organic) may influence the levels of antimicrobial resistance. By contrast,
for laying hens, because of the lack of epidemiological data on antimicrobial resistance, very
little is known about the influence of different housing systems on the occurrence of
antimicrobial resistance.
Therefore, the specific aims of this study were:
To determine the between- and within-flock prevalence of Salmonella in laying hen
flocks housed in conventional battery cages and non-cage housing systems.
To identify risk factors for the presence of Salmonella in laying hen flocks with
specific emphasis on the effect of the different housing systems
To determine the prevalence of antimicrobial resistance in indicator bacteria in laying
hens
To examine how the laying hen housing system influences the levels of antimicrobial
resistance
CHAPTER 3
FAECAL SAMPLING UNDER-ESTIMATES THE
ACTUAL PREVALENCE OF SALMONELLA IN
LAYING HEN FLOCKS
S. Van Hoorebeke, F. Van Immerseel, J. De Vylder, R. Ducatelle, F. Haesebrouck, F.
Pasmans, A. de Kruif and J. Dewulf
Adapted from Zoonoses and Public Health (2009) 56: 471-476
Chapter 3 61
ABSTRACT
In all EU member states, Salmonella monitoring in poultry flocks is obligatory. In
these monitoring programs a limited number of pooled faeces and / or dust samples are
collected to determine whether Salmonella is present in the flocks or not. Whether these
limited sampling protocols are sufficiently sensitive to detect expected low within-flock
prevalences of an intermittently shed pathogen is not yet clear. In this paper, a comparison is
made between different sampling procedures for the assessment of the between and within-
flock prevalence of Salmonella in laying hens. In total, 19 flocks were sampled. All the
sampled flocks were found negative for Salmonella based on the results from the official
Salmonella control program. Using an extended on-farm sampling methodology, Salmonella
could not be detected in any of the flocks. After transportation of the hens to the laboratory
and subsequent analysis of cloacal swabs and caecal contents, Salmonella Enteritidis was
detected in laying hens from 5 out of 19 farms. The observed within-flock prevalence ranged
from 1 – 14 %. Based on the results of this study, it can be expected that, depending on the
sampling procedure, different estimates of the prevalence of Salmonella can be obtained and
the proportion of Salmonella infected flocks is underestimated based on the results of the
official monitoring program.
Key words: Salmonella; laying hens; sampling; prevalence
62 Chapter 3
INTRODUCTION
In the European Union (EU), Salmonella is currently the second most important food-
borne pathogen (European Food Safety Authority, 2007). Outbreaks affecting only a few
individuals are most common but on some occasions outbreaks involving large numbers of
cases are observed (Bell and Kyriakides, 2002). Outbreaks of human salmonellosis in the EU
are predominantly caused by Salmonella Enteritidis and Typhimurium (European Food Safety
Authority, 2007). Contaminated poultry meat and eggs are the main sources of Salmonella
Enteritidis for humans (Davies and Breslin, 2001; Namata et al., 2007). Pork and pork-related
products are the main vectors for transfer of Salmonella Typhimurium to humans (Van Pelt et
al., 2000; Jansen et al., 2007). Since many years, Salmonella Enteritidis is the main cause of
human salmonellosis, both in Europe and in North-America, and this is mainly due to
consumption of contaminated eggs (Angulo and Swerdlow, 1998; Delmas et al., 2006; EFSA,
2006). Recently, a decrease in human Salmonella Enteritidis infections has been noted in
different countries (Cogan and Humphrey, 2003; Collard et al., 2008). This can most likely be
attributed to a number of measures that have been taken since the mid to late 1990s, such as
improved biosecurity and hygiene on breeder and laying hen farms, either or not in
combination with vaccination of commercial laying hens (EFSA, 2006; Mossong et al., 2006;
Wales et al., 2007).
The EU baseline study on the prevalence of Salmonella in laying hens, carried out in
2004-2005, has demonstrated a variable level of infection of laying hen holdings in the
different EU member states. The European Regulation No. 2160/2003 makes strict sampling
schemes mandatory in the EU member states in order to provide follow-up data on the level
of flock contamination: every flock of at least 1000 commercial laying hens has to be sampled
every 15 weeks with a first sampling at the age of 24 ± 2 weeks. The following samples need
to be collected during the production cycle: 2 pooled faeces samples of 150 g each (on farms
with a conventional battery cage or a furnished cage housing system) or 2 pairs of overshoes
(on farms with an aviary, floor-raised or free-range housing system) (Regulation No.
1168/2006). It is unclear however whether this sampling scheme is able to detect low
Salmonella contamination levels and therefore provides accurate follow-up data on the level
of laying hen holding contamination in the EU.
The aim of the study described below is to evaluate whether a more intensive sampling
methodology results in different estimates of the within and between flock prevalence in
commercial layer flocks.
Chapter 3 63
MATERIALS AND METHODS
Selection of the sampled farms
Flocks were selected based upon a list of contact addresses of registered laying hen
farms provided by the official Belgian Identification & Registration authorities. The only
inclusion criterion used was the flock size (>1000 hens). In total, 220 farms with a minimal
capacity of 1000 laying hens were registered. The farms which were in the last month of the
production cycle were contacted by telephone. Participation was voluntary. All of the
contacted farms volunteered to participate. In total 19 flocks from 19 different farms were
sampled, comprising conventional battery cage flocks and flocks housed in alternative
housing systems. On farms with more than one house, only one house was randomly selected
to be sampled. When several flocks were present in a given house, only one flock was
selected at random. The size of the selected flocks varied between 3500 and 29000 hens. All
sampled flocks were vaccinated against Salmonella and screened negative for Salmonella by
the official monitoring program which means that the flocks were sampled every 15 weeks
starting from 6 weeks after arrival of the hens on the farm (following EU Regulation No.
1168/2006).
Moment of sampling
Participating laying hen farms were sampled one week prior to depopulation. The age
of the sampled hens ranged from 70 to 82 weeks. The samplings were performed between
June 2007 and May 2008.
Number and sample type taken on-farm
On-farm sampling consisted of 5 pooled faeces samples, 1 mixed dust sample and 40
cloaca swabs of 40 randomly selected laying hens. Depending on the housing system, the
pooled faeces samples were taken as follows:
Caged flocks: 5 samples of mixed fresh faeces were taken from dropping belts,
scrapers or deep pits depending on the type of cage (conventional battery cage or
furnished cage). Each pooled faeces sample consisted of approximately 250 g.
Floor-raised, free-range and organic farms: for each of the 5 pooled faeces samples 60
piles of fresh faeces were collected from the floor and the slats. Each pooled faeces
sample consisted of approximately 250 g.
64 Chapter 3
The mixed dust sample was collected with a gloved hand on various places in the house
such as exhaust fan baffles, adjacent ledges, beams, partitions, pipes or underneath cages.
Enough dust was collected to fill a 250 ml jar. Both the pooled faeces samples and the mixed
dust sample were placed in a sterile recipient. Gloves were changed in between collection of
each pooled faeces sample and the mixed dust sample. The cloaca swabs were taken from
hens that could be caught without causing too much agitation. Care was taken that hens were
selected evenly throughout the house. Forty hens in the flock were selected and from each hen
a cloaca swab was taken by inserting a cotton-tipped swab approximately 5 cm into the cloaca,
taking care to avoid contact with the surrounding feathers and skin. Afterwards, the swabs
were placed in tubes containing Ames medium. The sample size of 40 swabs was calculated
in function of an expected within-flock prevalence of 20 %, an accepted error of 10 %, a
confidence level of 90 % and a flock size of 10000. All samples were placed in an appropriate
leak-proof bag and outer container and transported to the lab under ambient conditions and
were incubated for bacteriological analysis (see further) within 12 hours post-sampling.
From each flock, 100 hens were caught. The selection of 100 samples is based on the
formula used for the detection of disease (WinEpiscope 2.0). Based on this formula it is
shown that for the detection of a disease with a minimal prevalence of 3 % and a desired level
of confidence of detection of 95 % one needs a sample of 98 in a flock of 5000 animals or
more. The caught hens were placed in cleaned and disinfected transport boxes. Each transport
box was disinfected with a commercial available disinfectant proven to be effective against
Salmonella spp. (Virocid®). Separations of clean disposable cardboards prevented contact
between hens and faeces in the different boxes. The boxes containing the hens were
transported to the Faculty of Veterinary Medicine of Ghent University in a cleaned and
disinfected van. Transportation time ranged from 30 to 90 minutes. All possible hygienic
measures were taken during transportation of the hens to avoid contamination of the hens by
the environment: After each transport, the transport boxes were cleaned (first dry and
afterwards with water). Each transport box was disinfected with a commercial available
disinfectant proven to be effective against Salmonella spp. (Virocid®, CidLines, Belgium).
The van in which the hens were transported was also cleaned and disinfected. During
transport, clean disposable cardboards were used to avoid contamination of the hens of one
box with faeces of the hens of the neighboring boxes.
Chapter 3 65
Number and sample type taken after transport
Immediately after arrival at the Faculty of Veterinary Medicine, all hens were labeled
and a cloaca swab of each hen was taken in the same way as described above. After sampling,
each hen was euthanized by intravenous embutramid injection (T61®, Intervet, Belgium).
Subsequently, the hens were necropsied and both caeca were aseptically removed. Both caeca
of each hen were homogenized and pooled for further processing. Bacteriological analysis of
all samples started on the day of sampling.
Bacteriological analysis of samples
All samples were analyzed using a modification of ISO 6579:2002, as recommended
by the Community Reference Laboratory for Salmonella in Bilthoven, The Netherlands. From
each pooled faeces sample, 25 g was added to 225 ml of buffered peptone water (BPW)
(Oxoid, Basingstoke, Hampshire, UK). Of the mixed dust sample, there was twice 10 grams
of mixed dusty material separately added to 90 ml of BPW. Cloaca swabs were placed in 9 ml
of BPW. Pooled faeces samples and dust samples were mixed in a stomacher bag for 1 minute.
All samples were incubated for 18 ± 2 h at 37 ± 1 °C. Next, 3 droplets of the pre-enrichment
culture were inoculated onto a modified semi-solid Rappaport-Vassiliadis (MSRV) (Difco;
Becton Dickinson) agar plate containing 0.01 g l-1
novobiocine and incubated for 2 x 24 ± 2 h
at 42 ± 1°C. Suspect white culture from the border of the growth zone was plated on Brilliant
Green Agar (BGA; Oxoid) and Xylose Lysine Deoxycholate agar (XLD; Oxoid), followed by
incubation for 24 ± 2 h at 37 ± 1°C. Presumed Salmonella colonies on BGA and XLD were
biochemically confirmed using ureum agar, triple sugar iron agar and lysine-decarboxylase
broth. Serotyping of Salmonella-isolates according to the Kauffmann-White scheme was
performed at the Scientific Institute of Public Health (Brussels, Belgium).
Statistical analysis
Both for the between- as for the within-flock prevalence, 95 % confidence intervals
have been calculated using the formula for the calculation of confidence intervals (Thrusfield
M., 1995). The Pearson‟s χ2-test was used to determine significant differences between the
different sampling methods (P < 0,05).
66 Chapter 3
RESULTS
A detailed overview of the bacteriological analysis of samples is presented in Table 1.
Salmonella could not be detected in any of the pooled faeces samples and the mixed dust
samples. Salmonella could also not be detected using on-farm sampling of 40 individual hens
by taking cloaca swabs. After transportation of the animals, Salmonella was detected in laying
hens of 5 out of 19 farms, both in cloaca swabs and in the caeca. All of the isolated strains
belonged to the Salmonella Enteritidis serotype. Serotyping and antimicrobial susceptibility
testing was performed to make sure that it was not the live vaccine strain that was isolated.
In positive flocks, estimations of the within-flock prevalence differed depending on the
sample type. The within-flock prevalence determined using cloacal swabs was always below
4%, whereas the within-flock prevalence based on the bacteriological examination of the
caeca varied between 5 and 14 %. In all hens that were positive for Salmonella after
bacteriological analysis of cloaca swabs, the bacterium was also found in the caecal samples.
Table 1: Overview of the bacteriological analysis of samples from 19 laying hen flocks taken after
transport: number of positives per sample type (95% C.I.)
Farm Housing type Bacteriological analysis
Cloacal swabs after
transport
Caeca Phage types of S.
Enteritidis
1 Battery 3 (0 - 6.33) 6 (1.37 - 10.63) PT 1
2 Organic 0 0 -
3 Battery 0 0 -
4 Free-range 0 0 -
5 Floor-raised 0 0 -
6 Floor-raised 0 0 -
7 Floor-raised 3 (0 - 6.19) 10 (4.39 - 15.61) PT 21
8 Battery 1 (0 - 2.94) 14 (7.24 - 20.76) PT 12
9 Free-range 0 0 -
10 Free-range 4 (0.18 - 7.82) 7 (2.02 - 11.98) PT 1, PT 35
11 Organic 0 0 -
12 Free-range 0 0 -
13 Battery 0 0 -
14 Free-range 0 0 -
15 Battery 0 0 -
16 Floor-raised 0 0 -
17 Organic 0 0 -
18 Floor-raised 0 0 -
19 Organic 2 (0 - 4.74) 5 (0.74 - 9.26) PT 11
Chapter 3 67
DISCUSSION
Bacteriological analyses of faecal samples as requested in European Regulation No.
2160/2003 most likely under-estimate the actual prevalence of Salmonella in laying hen
flocks. Indeed, all 19 flocks were screened negative for Salmonella by the official monitoring
program, using analysis of faecal samples. Even the increased on-farm sampling, combining
bacteriological analysis of 40 cloaca swabs, 1 mixed dust sample and 5 pooled faeces samples,
did not result in the detection of Salmonella, although several studies have shown that
sampling of dust is a good method to detect a Salmonella infection on the farm (Davies and
Breslin, 2001; Mahé et al., 2008). After transportation, Salmonella Enteritidis was found in
laying hens of 5 farms. Both cloacal swabs and caecal homogenates were found positive in a
low number of sampled animals.
Based on analysis of these samples the between-flock prevalence can be estimated at
26.3 % (7.47 – 45.13). This is comparable to the between-holding prevalence estimate of
Salmonella Enteritidis for Belgium (27.7 %) determined during the EU-wide baseline study
performed in 2004-2005 at a time when the vast majority of laying hens in Belgium were not
vaccinated (EFSA, 2006). In the EU-wide baseline study a flock was identified as being
Salmonella positive if at least one out of the 5 pooled faeces or 2 dust samples was positive.
When using the same decision criterion in this study we did not find any flock positive which
would result in an estimated between-flock prevalence of 0 %. Also the estimates of the
within-flock prevalence in the positive flocks were largely depending upon the sample types
considered. The results of the analysis of individual cloaca swabs taken at the farm were all
negative, whereas in the infected flocks a limited number of cloacal swabs taken after
transport were positive (within-flock prevalence estimate never exceeded 4 %). In the flocks
where some cloaca swabs were found positive, analysis of caecal homogenates yielded more
Salmonella positive animals (estimated within-flock prevalence between 5 and 14 %). First it
should be taken in account that the differences between the results of the different sampling
procedures could be attributed to the fact that the number of samples taken after transport is
larger than the number of samples taken on the farm. Furthermore these differences may be
explained by the intermittent excretion of Salmonella by infected animals (Van Immerseel et
al., 2004) and the fact that stress, caused by the transport, may make hens go from a „carrier‟
state to a „shedding‟ state. It is well known that stress factors such as the onset of lay, high
temperatures, induced molting, final stages of the production period or transportation to the
slaughterhouse can cause recurrences of Salmonella excretion (Line et al., 1997; Humphrey,
68 Chapter 3
2006; van de Giessen et al., 2006; Golden et al., 2008). The results do suggest that individual
cloaca swabs taken at the farm, which was seen as a potential user-friendly way to obtain
some information about the within-flock prevalence in an infected flock, do not seem to be
appropriate to detect a low prevalence of infection. This is in accordance with what has been
found in the studies by Bichler et al. (1996) and Van Immerseel et al. (2004) where cloacal
swabs were taken from experimentally infected chickens.
Our data also suggest that on farms which are „apparently Salmonella-free‟, a
relatively large proportion of the hens may still carry the pathogen without shedding.
Therefore, the numbers of infected flocks based on the official monitoring programs are most
likely an underestimation of the true number of flocks in which Salmonella is still present.
The actual public health risk of the flocks, carrying low levels of Salmonella that are only
detectable using intensified sampling procedures is unclear. Indeed, one can argue that the
risk of egg contamination in these flocks probably is very low.
Defining a flock as positive based on the finding of at least one positive sample in
whatever bacteriological sampling methodology used is a very crude way of categorizing
flocks and does not take into account any estimation of the infection pressure in the flock. The
results of this study do suggest that, even in those flocks where Salmonella was found, the
infection pressure at the time of sampling was probably low. This is based on the observation
that in the conventional sampling methodology no positive samples were found (no indication
of active shedding) and only a limited number of shedders and carriers were found after
submitting the birds to transport stress and intensive sampling. This low infection pressure
can probably be attributed to the combination of vaccination and hygienic measures. The
observed results are in accordance with the results of several studies concerning the effect of
vaccination. Indeed it has been stated that vaccination, with the vaccines currently used in
Belgium, can reduce but not totally prevent the faecal shedding and systemic spread of
Salmonella Enteritidis (Van Immerseel et al., 2005; Gantois et al., 2006). This raises the
question whether the sampling methodology as it is currently used in the Salmonella
monitoring programs of several EU member states is accurate enough to detect Salmonella in
low prevalence flocks. Although postmortem examination of hens is labour-intensive and
expensive, the results of this study suggest that it is the best method for the detection of
Salmonella in low prevalence flocks (P < 0,05). It also permits to estimate the within-flock
prevalence and as a consequence, the risk of infection of humans through the consumption of
infected eggs since there is a good agreement between the level of caecal carriage and the
Chapter 3 69
prevalence of infected or contaminated eggs (Henzler et al., 1994, 1998; Schlosser et al., 1995;
Mallinson et al., 2000).
However, in the current study the possible infection of the hens during transport also
needs to be taken into account. Despite the relatively short transportation time and the
hygienic precautions taken, it cannot be completely ruled out that the laying hens were
infected during the transport from the farms to the Faculty since for broilers it has been
described that birds got infected with Salmonella during transport due to insufficiently
cleaned and disinfected transport crates and vehicles (Rigby et al., 1980; Rigby et al., 1982;
Heyndrickx et al., 2002).
In conclusion, it is clear that depending on the sampling procedure different estimates
of the between- and within-flock prevalence of Salmonella can be obtained. Analysis of faecal
samples clearly under-estimates the actual prevalence of Salmonella in laying hen flocks. The
results of this study can be a stimulus to pay further attention to the control and prevention of
Salmonella Enteritidis in commercial laying hen flocks.
ACKNOWLEDGEMENTS
This research was funded by the EU FP6, under the contract 065547 (Safehouse project). The
authors thank Sofie Haerens for technical assistance.
70 Chapter 3
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Visser G., 2006. Surveillance of Salmonella spp. and Campylobacter spp. in poultry
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Infection of Chickens Early Posthatch with a Low or high Dose of Salmonella enteritidis.
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and Van Duynhoven Y., 2000. Oorsprong, omvang en kosten van humane salmonellose,
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36: 187-197
CHAPTER 4
THE AGE OF PRODUCTION SYSTEM AND
PREVIOUS SALMONELLA INFECTIONS ON FARM
ARE RISK FACTORS FOR SALMONELLA
INFECTIONS IN LAYING HEN FLOCKS
S. Van Hoorebeke, F. Van Immerseel, J. De Vylder, R. Ducatelle, F. Haesebrouck, F.
Pasmans, A. de Kruif and J. Dewulf
Adapted from Poultry Science (2010) 89:1315-1319
Chapter 4 77
ABSTRACT
An explorative field study was carried out to determine risk factors for Salmonella
infections in commercial laying hen flocks. For this purpose twenty-nine laying hen flocks,
including farms using conventional and alternative housing systems, were intensively sampled
both on-farm and after transport as described in chapter 3. An on-farm questionnaire was used
to collect information on general management practices and specific characteristics of the
sampled flock such as flock size, age of the hens and age of the infrastructure. Salmonella was
detected in laying hens from 6 of the 29 sampled farms. Using multivariate logistic regression
with the Salmonella status of the flock as an outcome variable a previous Salmonella
contamination on the farm and the age of the production system were identified as risk factors
for Salmonella infections in laying hens (P < 0.05).
Key words: risk factor – Salmonella - laying hen
78 Chapter 4
INTRODUCTION
In the EU, Salmonella is still the second most important cause of food-borne
infections (EFSA, 2007). Although a decrease in human Salmonella Enteritidis infections has
recently been noted in a number of EU member states (Cogan and Humphrey, 2003; Collard
et al., 2007), contaminated eggs remain one of the most important sources of Salmonella
Enteritidis infections for humans (Delmas et al., 2006; EFSA, 2007). In order to reduce
Salmonella and other zoonotic agents of public health significance in farm animals, the EU
member states have to apply Regulation EC No 2160/2003 (Anon., 2003) into their national
legislation, which implies that EU member states have to invest in prevention, detection and
control of Salmonella infections in laying hens.
Only a few epidemiological studies have been carried out to investigate risk factors for
Salmonella infections in laying hen farms. The main risk factors that have been identified in
these studies are (1) large flock sizes (Mollenhorst et al., 2005; Namata et al., 2008) (2) the
age of the sampled hens, where a higher age implies a higher risk for Salmonella (Castellan et
al., 2004; Namata et al., 2008) and (3) the housing system, where conventional battery cages
showed a higher risk for Salmonella compared to alternative housing systems (EFSA, 2007).
However, it can be questioned whether the sampling methodologies that were used in
these studies (i.e. pooled faeces and dust samples (EFSA, 2007; Namata et al., 2008) or
serology (Mollenhorst et al., 2005)) are suitable for the detection of low Salmonella infection
levels, especially when these samplings are performed in flocks which have been vaccinated
against Salmonella. Moreover, in the EFSA baseline study it is clearly stated that the results
may have been confounded by farm size, flock size and other variables.
The aim of this paper is to describe the presence of Salmonella on 29 laying hen farms
using an extensive sampling protocol and to determine which management and farm
characteristics influence the Salmonella status of the flock.
MATERIALS AND METHODS
Selection of the Farms
Farms were selected using the National Identification & Registration database. There
are 220 laying hen farms in Belgium with a capacity of 1000 hens or more. In the target
population, the distribution of the farms is as follows: 60 % conventional battery cages (n =
132) and 40 % non-cage systems. Of these non-cage housing systems 40 % are floor-raised
farms (n = 37), 40 % are free-range (n = 33) and 20 % are organic systems (n = 18). To assure
Chapter 4 79
that the potential effect of the housing type on the Salmonella prevalence could be evaluated,
a stratified selection of the housing types was performed in order to keep the proportion of
conventional battery cages / non-cage systems approximately 1 / 4. Only farms with more
than 1000 laying hens that were in the last month of the production cycle were selected.
Owners of farms fulfilling these selection criteria were contacted by telephone. Participation
was voluntary.
Sample Types and Analysis of the Samples
In total 29 laying hen farms were sampled (8 conventional battery cage flocks, 10
floor-raised flocks, 8 free-range flocks and 3 organic flocks). Only one flock per farm was
sampled. All of the sampled flocks were vaccinated against Salmonella with an attenuated
vaccine. The following samples were collected on-farm: 5 pooled faeces samples, 1 mixed
dust sample and cloacal swabs of 40 randomly selected hens. Subsequently 100 randomly
selected hens per farm were transported to the Faculty of Veterinary Medicine. Of each hen a
cloacal swab was taken after transport. After euthanasia both caeca of each hen were removed
and pooled for further analysis. Detailed information on the bacteriological analysis of the
samples is described in Van Hoorebeke et al. (2009). Summarized, all samples were analyzed
using a modification of the ISO 6579:2002 method. Pre-enrichment was done by incubation
of the samples in buffered peptone water (Oxoid, Basingstoke) during 18 ± 2 h at 37 ± 1 °C.
Of the pre-enrichment solution 3 droplets were inoculated onto a modified semi-solid
Rappaport-Vassiliadis (MSRV) (Difco; Becton Dickinson) agar plate and incubation was
done for 2 x 24 ± 2 h at 42 ± 1°C. Suspect white culture from the border of the growth zone
was plated on Brilliant Green Agar (BGA; Oxoid) and Xylose Lysine Deoxycholate agar
(XLD; Oxoid), followed by incubation for 24 ± 2 h at 37 ± 1°C. Presumed Salmonella
colonies on BGA and XLD were biochemically confirmed using ureum agar, triple sugar iron
agar and lysine-decarboxylase broth. All sampled flocks were screened negative for
Salmonella by the official sampling protocols in accordance with EU regulation No
2160/2003.
Questionnaire Design
The questionnaire was filled in during an on-farm interview at the same day of sample
collection. Questions related to general farm and flock characteristics (e.g. flock size, breed,
age of the hens, medical treatments…) and biosecurity measures. Special attention was paid to
the housing system in which the sampled flock was housed (Table 1). The on-farm interview
took on average 25 minutes to complete.
80 Chapter 4
Data Processing and Analysis
Information from the questionnaires was coded and put in a database (Excel,
Microsoft Cooperation). Data were analyzed using SPSS 16.0 for Windows (SPSS Inc.,
Chicago, IL). The potential relationship between risk factors and Salmonella status of the
sampled farm was evaluated by means of a multivariate logistic regression model with the
Salmonella status of the sampled flock as a binary outcome variable. For this a flock was
defined infected if at least one of the collected samples was positive for Salmonella. Before
entering the variables into the multivariate model a univariate evaluation of each potential risk
factor was performed. All factors with a P-value < 0.20 were taken into account for the
multivariate model which was constructed in a stepwise backward manner. In the final
multivariate logistic regression model only factors with P < 0.05 were retained. All 2-way
Farm characteristics
Total capacity of the farm
Number of poultry houses
Other animal / poultry productions
Control of pests
House characteristics
Total capacity of the house
Size
Age of building and production system
Number of flocks present
Feeding / drinking / manure disposal systems
Access to out-door run
Nest boxes & egg collecting systems
Cleaning & disinfection status before repopulation
Biosecurity measures (footbath, clothing, all in/ all out)
Sampled flock characteristics
Number of hens
Age of the hens
Breed of the hens
Medical treatments
Salmonella vaccination status
Cumulative mortality in the flock since onset
Table 1: Summary of the main items included in the questionnaire to identify
risk factors for Salmonella in 29 laying farms (total number of questions = 92)
Chapter 4 81
interactions between significant main effects were tested. Odds ratios (OR), including 95 %
confidence intervals (CI), are reported for all significant variables.
RESULTS
A description of the main characteristics of the sampled farms is presented in Table 2.
The mean age of the hens at the moment of sampling was 75.72 weeks (min 68 weeks - max
82 weeks). No significant differences in age of the hens could be observed in the different
housing systems. The age of the production system was defined as the number of years that
the current infrastructure is in use. For conventional battery cages this was the number of
years since the current cages were installed. For alternative housing systems this was the
number of years since the current equipment (nest boxes, perches, slats…) was installed in the
sampled house. For this characteristic a remarkable difference was seen between the housing
systems (Table 2). The age of the production system was significantly higher in conventional
battery cage and organic farms compared to floor-raised and free-range farms (P = 0.01).
With regard to the length of the interval between depopulation and repopulation, a significant
longer interval could be observed in the alternative housing systems than in the conventional
battery cages (P < 0.05).
Table 2: Description of the main characteristics of the 29 sampled farms
Mean 95 % CI of the
mean
S.D. Minimum Maximum
Number of hens on the farm
Battery 30375 9887.3-50862.7 24506.2 9000 72000
Floor-raised 23400 18487.7-28312.4 6867.0 19000 38000
Free-range 23500 13290.2-33709.8 12212.4 4000 40000
Organic 5500 1021.7-9978.3 1802.8 3500 7000
Mean age of the production
system (years)
Battery 14.50 10.50-18.50 4.78 8.00 22.00
Floor-raised 5.60 3.60-7.60 2.80 1.00 10.00
Free-range 9.12 3.57-14.68 6.64 2.00 19.00
Organic 16.33 2.65-30.01 5.51 10.00 20.00
Interval depop-repop (days)*
Battery 20.12 12.19-28.06 9.49 14.00 42.00
Floor-raised 30.50 19.96-41.04 14.73 14.00 70.00
Free-range 31.25 22.22-40.28 10.81 21.00 56.00
Organic 35.00 4.88-65.12 12.12 21.00 42.00
*this is the number of days between the end of the previous production cycle and the restocking of the house with new laying hens (all farms all-in/ all-out)
82 Chapter 4
None of the 29 farms were found positive in any of the samples taken on-farm.
However, Salmonella could be detected in hens from 6 out of the 29 laying hen farms both in
the caeca and in the cloacal swabs taken after transport. A detailed overview of the results of
the bacteriological analysis of the 6 positive farms is presented in Table 3. The factors
associated with detection of Salmonella in the univariate analysis are listed in Table 4. The
housing system as such was not significantly associated with the Salmonella infection (P =
0.83) since the same ratio of positive farms was found in each category of housing systems.
Table 3: Results of bacteriological analysis of samples from laying hens of 29 flocks, after transport
Farm Housing type Cloacal swabs Caeca Serotype-phage type
1 Battery 3/100 6/100 Enteritidis, PT 1
2 Battery 1/100 14/100 Enteritidis, PT 12
3 Floor-raised 3/100 10/100 Enteritidis, PT 21
4 Free-range 4/100 7/100 Enteritidis, PT 1 and PT 35
5 Free-range 2/100 5/100 Enteritidis, PT 11
6 Organic 2/100 8/100 Typhimurium var. Copenhagen, PT 208
In the final multivariate logistic regression model (Table 5) the age of the production
system (P = 0.04) and a previous Salmonella contamination on the farm (P = 0.03) turned out
to be risk factors for a Salmonella-infection in laying hen flocks.
Table 4: Results of univariable analysis for the identification of risk factors for Salmonella infection
on 29 laying hen farms. Only factors with a P-value < 0.2 are listed.
Continuous variables N OR 95 % CI for OR P-value
Age of the production system 29 1.29 1.05-1.57 0.02
Categorical variable Salm pos/n OR 95% CI for OR P-value
Previous Salmonella-contamination
No (ref) 2/24 - - -
Yes 4/5 44.00 3.18-608.16 < 0.01
Other animal production on the farm
No (ref) 1/20 - - -
Yes 5/9 23.75 2.15-262.47 0.01
Sanitary transition zone present
No 3/5 10.50 1.21-91.03 0.03
Yes (ref) 3/24 - - -
Type of ventilation
Natural 2/3 11.00 0.80-152.04 0.07
Mechanical (ref) 4/26 - - -
Control of rodents 0.18
By farmer (ref) 3/22 - - -
By farmer and specialized company ¼ 2.11 0.16-27.58 0.56
By specialized company 2/3 12.67 0.86-186.91 0.06
Chapter 4 83
Table 5: Results of multivariable analysis for the identification of risk factors for Salmonella infection
on 29 laying hen farms (P-value < 0.05)
Variable n OR 95 % CI for OR P-value
Age of the production system 29 1,35 1,01-1,81 0.04
Previous Salmonella contamination
No (Ref.) 24 - - -
Yes 5 77.64 1.68-3596.30 0.03
DISCUSSION
The number of sampled flocks in this study is relatively small which limits its power
in comparison to the European baseline study on the prevalence of Salmonella in egg laying
flocks. Also there is the fact that the numbers of farms of the different housing types sampled
in this study do not exactly reflect the distribution of farms in the target population. This
might explain why in contrast to what has been found in the EU baseline study (EFSA, 2007;
Namata et al., 2008), the housing of the hens in conventional battery cages could not be
identified as a risk factor for Salmonella in this dataset. It should be stressed that if the same
sampling methodology as in the EU baseline study (i.e. 5 pooled faeces samples and 2 dust
samples) was used no positive flock would have been detected. This indicates that the
infection level in the positive flocks was very low. These low level infections were, at least in
the subset of sampled flocks, present in equal proportions in the different housing systems.
This could suggest that for these low infection levels the housing system has no influence,
although more studies using extensive sampling should be performed to confirm this.
The fact that a previous Salmonella infection in the same house or in other houses on
the farms could be identified as a risk factor is in accordance with Huneau-Salaün et al.
(2009). Carrique-Mas et al. (2009) stated that the presence of Salmonella (Enteritidis) in the
farm environment plays an important role in the presence of Salmonella in a laying hen flock.
In all housing systems, poor standards of cleaning of the hens‟ houses could be observed in
many of the sampled flocks, especially in the sanitary transition zones and the anterooms of
the laying hens‟ houses. Similar poor results in biosecurity on laying hen farms have been
reported by Davies and Breslin (2003) and Wales et al. (2006). It has indeed been stated that
many Salmonella infections originate from the farm environment (van de Giessen et al., 1994;
Davies and Breslin, 2003). Moreover, the level of environmental contamination increases
84 Chapter 4
significantly during a production cycle (Wales et al., 2007). Salmonella can be detected both
on the floor as on the equipment at different locations in and around laying hen houses such as
underneath cages, the interior of the nest boxes, in laying house anterooms and egg storage
areas (Davies and Breslin, 2001; Davies and Breslin, 2003). In this study, cleaning and
disinfection during and between 2 production cycles of the above mentioned spots was
marginal or non existent on many of the sampled farms.
Furthermore the infection of a flock with Salmonella through vectors such as rodents,
flies and beetles (Davies and Breslin, 2001; Carrique-Mass et al., 2009) and the reported
capacity of some Salmonella strains isolated in poultry to develop a biofilm could contribute
to the survival of Salmonella in the environment of poultry houses (Marin et al., 2009).
Independent of the housing system, a higher age of the production system increased
the risk of presence of Salmonella on the farm. The significant longer interval between
depopulation and repopulation (P-value < 0,05) in alternative housing systems compared to
conventional battery cages does not seem to have a protective influence on the prevalence of
Salmonella. This is in accordance with Davies and Wray (1996) who described survival of
Salmonella in empty poultry houses for a period of 12 months.
A previous Salmonella contamination on the farm and the age of the production
system were found to be the main risk factors for low-level Salmonella infections occurring in
laying hen flocks, irrespective of the housing system in which the hens are housed. This
indicates that in flocks which are vaccinated against Salmonella, persistent biosecurity
measures are necessary in all types of housing systems in order to prevent the recurrent
contamination or new infections of laying hen flocks in subsequent production cycles.
ACKNOWLEDGEMENTS
This research was funded by the EU FP6, under contract 035547 (Safehouse project).
Chapter 4 85
REFERENCES
Anonymous, 2003. Regulation (EC) No 2160/2003 of the European Parliament and of the
Council of 17 November 2003 on the control of salmonella and other specified food-borne
zoonotic agents. Official Journal of the European Union 2003 L 325/1: 12.12.2003
Castellan D.M., Kinde H., Kass P.H., Cutler G., Breitmeyer R.E., Bell D.D., Ernst R.A.,
Kerr D., Little H.E., Willoughby D., Riemann H., Ardans A., Snowdon J.A. and Kuney D.R.,
2004. Descriptive study of California egg layer premises and analysis of risk factor for
Salmonella enterica serotype enteritidis as characterized by manure drag swabs. Avian Dis.
48: 550-561
Carrique-Mas J.J. and Davies R. H., 2008. Salmonella Enteritidis in commercial layer flocks
in Europe: Legislative background, on-farm sampling and main challenges. Braz. J. Poult. Sci.
10: 1-9
Carrique-Mas J.J., Breslin M., Snow L., Mc Laren I., Sayers A.R. and Davies R.H., 2009.
Persistence and clearance of different Salmonella serovars in buildings housing laying hens.
Epidemiol. Inf. 137: 837-846
Cogan T.A. and Humphrey T.J., 2003. The rise and fall of Salmonella Enteritidis in the UK. J.
Appl. Microbiol. 94: 114S-119S
Collard J.M., Bertrand S., Dierick K., Godard C., Wildemauwe C., Vermeersch K., Duculot J.,
Van Immerseel F., Pasmans F., Imberechts H. and Quinet C., 2008. Drastic decrease of
Salmonella Enteritidis isolated from humans in Belgium in 2005, shift in phage types and
influence on foodbourne outbreaks. Epidemiol. Inf. 136: 771-781
Davies R. and Wray C., 1996. Persistance of Salmonella enteritidis in poultry units and
poultry food. Br. Poult. Sci. 37: 589-596
Davies R. and Breslin M., 2001. Environmental contamination and detection of Salmonella
enterica serovar enteritidis in laying flocks. Vet. Rec. 149: 699-704
Davies R. and Breslin M., 2003. Observations on Salmonella contamination of commercial
laying farms before and after cleaning and disinfection. Vet. Rec. 152: 283-287
Delmas G., Gallay A., Espié E., Haeghebaert S., Pihier N., Weill F.X., De Valk H., Vaillant V.
and Désenclos J.C., 2006. Foodborne-diseases outbreaks in France between 1996 and 2005.
B.E.H. 51/52: 418-422
European Food Safety Authority (2007). Report of the Task Force on Zoonoses Data
Collection on the Analysis of the baseline study on the prevalence of Salmonella in holdings
of laying hen flocks of Gallus gallus, EFSA J. 97 84 pp.
Guard-Petter J. (2001). The chicken, the egg and Salmonella enteritidis. App. Envir.
Microbiol. 3: 421-430
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Huneau-Salaün A., Chemaly M., Le Bouquin S., Lalande F., Petetin I., Rouxel S., Michel V.,
Fravallo P. and Rose N., 2009. Risk factors for Salmonella enterica subsp. enterica
contamination in 519 French laying hen flocks at the end of the laying period. Prev. Vet. Med.
89: 51-58
Marin C., Hernandiz A. and Lainez M., 2009. Biofilm development capacity of Salmonella
strains isolated in poultry, risk factors and their resistance against disinfectants. Poult. Sci. 88:
424-431
Mollenhorst H., van Woudenbergh C.J., Bokkers E.G.M. and de Boer I.J.M., 2005. Risk
factors for Salmonella Enteritidis infections in laying hens. Poult. Sci. 84: 1308-1313
Namata H., Méroc E., Aerts M., Faes C., Cortinas Abrahantes J., Imberechts H. and Mintiens
K., 2008. Salmonella in Belgian laying hens: an identification of risk factors. Prev. Vet. Med.
83: 323-336
van de Giessen A.W., Ament A.J.H.A. and Notermans S.H.W., 1994. Intervention strategies
for Salmonella enteritidis in poultry flocks: a basic approach. Int. J. Food. Microbiol. 21: 145-
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Van Hoorebeke S., Van Immerseel F., De Vylder J., Ducatelle R., Haesebrouck F., Pasmans
F., de Kruif A. and Dewulf J., 2009. Faecal sampling underestimates the actual prevalence of
Salmonella in laying hen flocks. Zoonoses Public Health 56: 471-476
Wales A., Breslin M. and Davies R., 2006. Assessment of cleaning and disinfection in
Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture
techniques. Vet. Microbiol. 116: 283-293
Wales A., Breslin M., Carter B., Sayers R. and Davies R., 2007. A longitudinal study of
environmental salmonella contamination in caged and free-range layer flocks. Avian Path. 36:
187-197
CHAPTER 5
DETERMINATION OF THE WITHIN- AND
BETWEEN-FLOCK PREVALENCE AND
IDENTIFICATION OF RISK FACTORS FOR
SALMONELLA SHEDDING IN LAYING HEN
FLOCKS
S. Van Hoorebeke, F. Van Immerseel, J. Schulz, J. Hartung, M. Harisberger, L. Barco, A.
Ricci, G. Theodoropoulos, E. Xylouri, J. De Vylder, R. Ducatelle, F. Haesebrouck, F.
Pasmans, A. de Kruif and J. Dewulf
Adapted from Preventive Veterinary Medicine (2010) 94: 94-100
Chapter 5 91
ABSTRACT
Salmonella outbreaks in humans are often linked with the consumption of
contaminated eggs. Therefore a profound knowledge of the actual prevalence of Salmonella
spp. in laying hens and the factors that influence the presence and persistence of Salmonella
on a farm is of utmost importance. The housing of laying hens in conventional battery cages
will be forbidden in the European Union (EU) from 2012 onwards. There is an urgent need to
evaluate whether this move to alternative housing systems will influence the prevalence of
Salmonella in laying hens. Therefore, a cross-sectional study was performed in 5 European
countries (Belgium, Germany, Greece, Italy and Switzerland) to determine the between and
within flock prevalence of hens shedding Salmonella and to investigate whether there is an
effect of the housing type on the shedding of Salmonella. In total 292 laying hen farms were
sampled in the month prior to depopulation. The on-farm questionnaire described in chapter 4
was used to collect information on general management practices and specific characteristics
of the sampled flock. Twenty-nine flocks were found positive for at least 1 Salmonella-
serotype. In these flocks the within flock prevalence of shedding hens, determined by
individual sampling of 40 hens, varied between 0 % and 27.50 %. A wide variety of serotypes
was isolated with Salmonella Enteritidis as the most common. Housing in conventional
battery cages, the absence of dry cleaning in between production rounds and sampling in
winter turned out to be risk factors for the shedding of Salmonella Enteritidis or Typhimurium
(P < 0.05).
Key words: Salmonella – laying hens – prevalence – risk factors – housing
92 Chapter 5
INTRODUCTION
Already since the late 1960‟s there was a growing public awareness concerning farm
animal welfare which generally resulted in a consumer‟s aversion to eggs produced by laying
hens housed in cages (Appleby, 2003). This finally led to a ban of the housing of laying hens
in conventional battery cages in the EU, from January 1st 2012 onwards (Council Directive
1999/74/EC). From this date on, the housing of laying hens in the EU will be restricted to
enriched cages and alternative systems. According to the legislation laying hens housed in
enriched cages must have at least 750 cm2 of floor space per hen, a nest, perches and litter.
These enriched cages exist in a wide variety of group sizes (EFSA, 2005). The so-called
alternative systems are non-cage systems consisting of an indoor area either or not combined
with outdoor facilities (EFSA, 2005; LayWel, 2006). The outdoor run may be covered
(„wintergarden‟) or uncovered („free-range‟). Two main categories can be distinguished:
single level systems where the ground floor is fully or partially covered with litter and aviaries
with a ground floor area plus one or more platforms (EFSA, 2005; LayWel, 2006). The above
mentioned ban on conventional battery cages aims to improve the welfare of laying hens
(Wall et al., 2004). Yet it has also initiated the question whether there aren‟t any adverse
consequences of this decision on the spread and/or persistence of infectious diseases in a flock.
This fear is based on the opinion that one of the big advantages of conventional battery cages
is that, because hens are separated from their faeces, the risk for disease transmission through
faeces can be minimized (Duncan, 2000). As an example, in Switzerland an increase in the
incidence of bacterial infections in chicks and laying hens could be seen in a 12 year period
after the Swiss ban of conventional battery cages (Kaufmann-Bart and Hoop, 2009). This
leads to the question whether the same effect is to be expected for zoonotic pathogens such as
Salmonella or Campylobacter. After all Salmonella is worldwide still an important cause of
human disease (EFSA, 2009). In Europe, Salmonella Enteritidis and Salmonella
Typhimurium are the most commonly isolated serotypes in human cases of salmonellosis
(WHO, 2006; EFSA, 2007) and contaminated eggs still remain the most important source of
infection with Salmonella Enteritidis for humans (Crespo et al., 2005; De Jong and Ekdahl,
2006; Delmas et al., 2006).
The EU ban on conventional battery cages combined with high EU Salmonella
prevalence data, indicated by the results of the European Food Safety Authority (EFSA)
baseline study on the prevalence of Salmonella in European laying hen flocks (EFSA, 2007)
urged the European Commission to call for a specific targeted research project to analyze and
Chapter 5 93
control egg contamination by Salmonella after the move of laying hens to enriched cages and
alternative housing systems (the Safehouse-project). One of the main tasks was a cross-
sectional field study carried out in 5 European countries (Belgium, Germany, Greece, Italy
and Switzerland).
The aim of this paper was to determine the between and within flock prevalence of
Salmonella spp. in laying hen flocks housed in conventional battery cage and alternative non-
cage housing systems and to identify risk factors for the shedding of Salmonella on laying hen
farms.
MATERIAL AND METHODS
Selection of the sampled farms
Flocks were selected based upon a list of contact addresses of registered laying hen
farms provided by the official Identification & Registration authorities. Only farms with a
farm size of >1000 hens were selected. Also the housing system was an important selection
criterion. The composition of the subset of sampled farms that was aimed at was 1/5
conventional battery cage farms and 4/5 non-cage housing systems. Within these non-cage
housing systems an equal presence of aviaries, floor-raised, free-range and organic farms was
strived at.
The farmers were contacted by telephone. The purpose of the study was explained and
100% anonymity was guaranteed (except for Germany), the foreseen date of depopulation
was marked to make sure that the farm could be sampled in the month prior to depopulation.
The aim was to sample in total 340 farms. Since not all of the contacted farmers volunteered
to participate and because of logistic reasons, this number was not entirely reached. In the
final study 292 flocks from 292 different farms were sampled in Belgium (n = 69), Germany
(n = 84), Greece (n = 10), Italy (n = 30) and Switzerland (n = 99), comprising conventional
battery cage flocks and four types of non-battery cage housing systems (Table 1). On farms
with more than one house, only one house was randomly selected to be sampled. When
several flocks were present in a given house, only one flock was selected at random.
Moment of sampling
Participating laying hen farms were sampled one month prior to depopulation. The
samplings were performed during a 19-month period from January 2007 to August 2008.
94 Chapter 5
Table 1: Numbers and types of laying hen farms that have been sampled
Country Conventional
battery cage
Floor
raised
Free range Organic Aviary Total
Belgium 18 21 21 8 1 69
Germany 26 20 24 14 - 84
Italy 12 10 8 - - 30
Greece 3 7 - - - 10
Switzerland - 16 33 14 36 99
Total 59 74 86 36 37 292
Number and sample type taken on-farm
On-farm sampling consisted of 5 pooled faeces samples, 40 cloaca swabs of 40 laying
hens and 1 sample of 200 red mites. Depending on the housing system, the pooled faeces
samples were taken as follows:
Conventional battery cage flocks and aviaries: 5 samples of mixed fresh faeces were
taken from dropping belts and scrapers. The farmer was asked to operate the manure
belts of each stack. In this way fresh piles of faeces could be picked up from each
level of manure belts and of cages both at the right and the left side of the stack and
along the entire length of stack. Each pooled faeces sample consisted of approximately
250 g.
Floor-raised, free-range and organic farms: for each of the 5 pooled faeces samples 60
piles of fresh faeces were collected from the floor and the slats. Each pooled faeces
sample consisted of approximately 250 g.
The pooled faeces samples were placed in a sterile recipient. Gloves were changed in
between collection of each pooled faeces sample. The red mites were collected wherever in
the house they were available. Using a disinfected brush they were swept into a sterile
recipient.
The cloaca swabs were taken from hens that could be caught without causing too much
agitation. Care was taken that hens were selected evenly throughout the house. Forty hens in
the flock were selected and from each hen a cloaca swab was taken by inserting a cotton-
tipped swab approximately 5 cm into the cloaca, taking care to avoid contact with the
surrounding feathers and skin. Afterwards, the swabs were placed in tubes containing Ames
medium. The sample size of 40 swabs was calculated in function of an expected within-flock
prevalence of 10 %, an accepted error of 10 %, a confidence level of 95 % and a flock size of
10000 (WinEpiscope 2.0). All samples were placed in an appropriate leak-proof bag and outer
Chapter 5 95
container and transported to the lab under ambient conditions and were incubated for
bacteriological analysis (see further). Bacteriological analysis started on the day of sampling.
If not, samples were stored at 4°C for no more than 24 hours.
Bacteriological analysis of samples
All samples were analyzed using ISO 6579:2002_Amd1:2007, as recommended by the
Community Reference Laboratory for Salmonella in Bilthoven, The Netherlands for the
detection of Salmonella in animal and environmental samples from primary production. From
each pooled faeces sample, 25 g was added to 225 ml of buffered peptone water (BPW)
(Oxoid, Basingstoke, Hampshire, UK). Each cloacal swab was placed in 9 ml of BPW. Pooled
faeces samples were mixed in a stomacher bag for 1 minute. All samples were incubated for
18 ± 2 h at 37 ± 1 °C. Next, 3 droplets of the pre-enrichment culture were inoculated onto a
modified semi-solid Rappaport-Vassiliadis (MSRV) (Difco; Becton Dickinson) agar plate
containing 0.01 g l-1
novobiocine and incubated for 2 x 24 ± 3 h at 41.5 ± 1°C. Suspect white
culture from the border of the growth zone was plated on Brilliant Green Agar (BGA; Oxoid)
and Xylose Lysine Deoxycholate agar (XLD; Oxoid), followed by incubation for 24 ± 3 h at
37 ± 1°C. Presumed Salmonella colonies on BGA and XLD were biochemically confirmed
using ureum agar, triple sugar iron agar and lysine-decarboxylase broth. Serotyping of
Salmonella-isolates according to the Kauffmann-White scheme was performed in the national
Reference Labs for Salmonella in each participating country.
Questionnaire design
The questionnaire was filled in during an on-farm interview on the same occasion of
the collection of the samples. Questions related to general farm and flock characteristics (e.g.
flock size, breed, age of the hens, medical treatments…) and biosecurity measures. Special
attention was paid to the system in which the sampled flock was housed. A summary of the
main items included in the questionnaire is given in Table 2. The same questionnaire was
used in all participating countries. Before use the questionnaire was tested on 2 laying hen
farms, one with conventional battery cages and one with a free-range production system to
check whether the questions were relevant to the aim of this study.
96 Chapter 5
Data processing and analysis
Information from the questionnaires was coded and put in a database (Excel,
Microsoft Cooperation). Data were analyzed using SPSS 16.0 for Windows (SPSS Inc.,
Chicago, IL) and MLwiN (MLwiN version 2.0, Centre for Multilevel Modelling, Institute of
Education, London, UK). One-way analysis of variance was used to examine differences in
farm and flock size and age of the infrastructure between the different housing systems. For
conventional battery cages this was the number of years since the current cages were installed.
For the other housing systems this was the number of years since the current equipment (nest
boxes, perches, slats…) was installed in the sampled house.
Table 2: Summary of the main items included in the questionnaire to identify risk factors for Salmonella in laying hen farms (for continuous variables: Median; for categorical variables:
proportions) (total number of questions = 92)
All flocks
Salm.*
positive
flocks
Salm.*
negative
flocks
Farm characteristics
Total number of hens 9700.0 57500.0 9000.0
Number of poultry houses 1.0 1.5 1.0
Other animal production present (% of the farms) 48.3 22.7 50.4
Control of pests (% of the farms) 91.0 90.1 91.1
House and management characteristics
Age of building (in years) 21.0 38.0 19.0
Age of infrastructure 9.0 21.0 9.0
Number of flocks present 1.0 1.5 1.0
Access to out-door run (% of the farms) 63.4 22.3 66.7
Number of egg collections per day 2.0 1.0 2.0
Dry cleaning status (% of the flocks) 82.2 31.8 86.3
Sampled flock characteristics
Number of hens in the flock 4820 22000.0 4798.0
Cumulative mortality in the flock since onset (in %) 8.00 7.00 8.00
Age of the hens (in weeks) 71.0 73.0 71.0
Salmonella vaccination status (% of the flocks) 28.1 13.6 29.3
Medical treatment (% of the flocks) 9.2 18.2 8.5
*Salmonella Enteritidis and/or Typhimurium
The within flock prevalence of excretion for each flock positive for Salmonella
Enteritidis or Typhimurium was calculated based on the 40 individual cloacal swabs. The 95 %
confidence interval (CI) was calculated as the prevalence ± 1.96 SD/√n with SD being the
standard deviation and n the sample size. For positive flocks with 0 positive cloaca swabs
(only positive in pooled faeces) the maximum possible prevalence that could be missed due to
Chapter 5 97
coincidence was calculated using following formula:
(WinEpiscope 2.0). With D = maximum number of positive animals missed due to
coincidence, CL = the confidence level, N = population size and n = sample size.
The relationship between risk factors and Salmonella Enteritidis or Typhimurium
status of the sampled farm was evaluated by means of a multilevel logistic regression model
with the Salmonella Enteritidis/Typhimurium status of the sampled flock as a binary outcome
variable. For this a flock was defined as being infected if at least one of the collected samples
was positive for Salmonella Enteritidis or Typhimurium. The decision to take this outcome
variable is based on the fact that these 2 serovars are the 2 most common isolated serovars in
case of human salmonellosis (EFSA, 2009). To check the potential effect of the housing
system, the following categories were used: conventional battery cages, indoor production
(which means aviaries and floor-raised systems), free-range systems and free-range organic
systems.
In a first step, all potential risk factors were tested univariably and only variables with
a P-value < 0.2 were selected. The shape of the relationship with the outcome variable was
assessed for all continuous variables by plotting the log odds of the outcome versus the
continuous variable (Parkin et al., 2005). If there was a non-linear relationship, the continuous
variable was categorized.
Next, a multivariable logistic regression model was built, including all variables with a
univariable P-value < 0.20. Correlations between the selected independent variables
(Pearson‟s and Spearman‟s ρ correlations) were determined. If 2 variables were highly
correlated (this means with an r2-value above 0.60), only the variable with the smallest P-
value was included in the final multivariable model.
The distribution of the housing types within the different countries could possibly
influence the risk factor analysis, because of clustering or non-independence of data. To
address this, the analyses were performed using MLwiN. Hereby, „country‟ was taken into
account as a random effect. To evaluate the presence of confounding, a Mantel-Haensel
analysis was used. Confounding was considered potentially significant when changes in the
OR of more than 20 % could be observed (Dohoo et al., 2003).
In the final multilevel logistic regression model, all two-way interactions between
significant variables were evaluated (with the significance level set at P < 0.05). Odds ratios
(OR), including 95 % confidence intervals (CI), are reported for all significant variables.
98 Chapter 5
RESULTS
A total of 292 commercial laying hen farms have been sampled in Belgium, Germany,
Greece, Italy and Switzerland. A detailed overview of the numbers and types of farms is
presented in Table 1. The participation rate was more than 90 % in Belgium, Greece, Italy and
Switzerland and 70 % in Germany. A description of the main characteristics of the sampled
farms is presented in Table 3. The mean age of the sampled hens was 72.1 weeks, ranging
from 45 to 121 weeks. Because of practical reasons 1 Greek flock was sampled halfway the
production cycle, at 38 weeks of age. The size of the selected flocks varied between 480 and
96000 hens. The number of laying hens on conventional battery cage farms was significantly
higher than the number of hens on farms with a non-cage housing system (P < 0.05). The age
of the infrastructure in conventional battery cage systems was significantly higher than on
floor-raised, free-range and organic farms (P < 0.05). No significant difference in the number
of houses on the farm could be seen between the different housing systems, neither was there
a significant difference in the number and type of sampled houses between the seasons.
Table 3: Main characteristics of the 292 sampled farms
Median St. Dev. Minimum Maximum
Amount of hens on the farm
Battery 55500 88360.6 2500 540000
Aviary 4996 4849.9 1760 23600
Floor-raised 13100 48333.7 1000 304000
Free-range 9000 15002.8 1000 80000
Organic 2845 2916.5 1000 12000
Amount of hens in the flock
Battery 24300 18394.6 1000 96000
Aviary 2537 1785.3 480 10242
Floor-raised 6500 5652.4 950 28500
Free-range 4361 6133.1 1000 28000
Organic 1862 1820.4 1000 7000
Age of the production system (years)
Battery 14.0 12.0 1.0 46.0
Aviary 16.0 7.1 1.0 33.0
Floor-raised 8.0 10.5 1.0 45.0
Free-range 7.0 7.9 1.0 43.0
Organic 8.0 5.3 1.0 20.0
Age hens (weeks)
Battery 74.0 11.6 45 121
Aviary 67.0 9.8 60 97
Floor-raised 70.0 10.7 38 112
Free-range 70.0 7.0 49 90
Organic 68.0 6.1 62 84
Chapter 5 99
Of the 292 sampled farms, 29 were found positive for Salmonella in at least one of the
samples taken. The between flock prevalences differed significantly (P < 0.05) between the
countries: 1.43 % (0.00 – 3.73 %) (Belgium), 20.00 % (11.68 – 28.72 %) (Germany), 20.00 %
(0.00 – 44.58 %) (Greece) and 30.00 % (13.81 – 46.19 %) (Italy). In none of the 99 sampled
Swiss farms Salmonella could be found: 0.00 % (0.00 – 3.70 %). When looking only at
Salmonella Enteritidis or Typhimurium, 22 flocks were found positive with respective
between flock prevalences of 1.43 % (0.00 – 3.73 %) (Belgium), 20.00 % (11.68 – 28.72 %)
(Germany), 0.00 % (0.00 – 2.40 %) (Greece) and 13.30 % (1.32 – 25.34 %) (Italy).
Only 7 of the 29 positive farms were found positive both in the pooled faeces samples
and in the cloacal swabs. Twenty flocks were only positive in the pooled faeces samples and 2
flocks were positive in one or more cloacal swabs but not in the pooled faeces samples. The
number of positive pooled faeces samples ranged from 1 to 5. In the 9 flocks where positive
cloacal swabs were found the within-flock prevalence varied between 2.50 % and 27.50 %. In
the 20 flocks with positive pooled faeces but no positive cloacal swabs the maximum
prevalence of hens shedding Salmonella that could have been missed due to coincidence was
7.33 %. A detailed overview of the results of the bacteriological analysis with the within flock
prevalences is presented in Table 4. The number of positive swabs was not significantly
associated with the housing type, nor was the number of positive pooled faeces samples. On
214 of the 292 sampled farms red mites could be collected. None of the red mites samples
was found positive for Salmonella.
100 Chapter 5
Table 4: Detailed overview of the 29 laying hen flocks found positive for Salmonella
Country Housing
type
N positive / Sample
type
Within flock
prevalence (95 % CI)
Serotype and phage type
Belgium Conv. Batt. 5/5 faeces,0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 4a
Germany Conv. Batt. 5/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 21c, RDNC
Germany Conv. Batt. 3/5 faeces ,0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 4
Germany Conv. Batt. 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 4
Germany Conv. Batt. 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 4
Germany Conv. Batt. 1/5 faeces, 2/40 swabs 5.00 % (0.00 – 11.65 %) S. Enteritidis PT 8
Germany Conv. Batt. 1/5 faeces,0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 4
Germany Conv. Batt. 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 4
Germany Conv. Batt. 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.18 %) S. Enteritidis PT 8
Germany Conv. Batt. 2/5 faeces, 1/40 swabs 2.50 % (0.00 – 7.33 %) S. Enteritidis PT 4
Germany Conv. Batt. 2/5 faeces, 0/40 swabs 0 % (0.00 – 7.33 %) S. Enteritidis PT 4
Germany Floor-raised 3/5 faeces, 4/40 swabs 10.00 % (0.73 – 19.27 %) S. Enteritidis PT 4
Germany Floor-raised 3/5 faeces, 1/40 swabs 2.50 % (0.00 – 7.33 %) S. Enteritidis PT 8
Germany Floor-raised 3/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 8, S. subsp. I
Germany Free-range 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.10 %) S. Enteritidis PT 4
Germany Free-range 5/5 faeces, 11/40 swabs 27.5 % (13.94 – 41.06 %) S. Enteritidis PT 4
Germany Free-range 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.18 %) S. Enteritidis PT 4
Germany Organic 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.16 %) S. Enteritidis PT 4
Greece Floor-raised 5/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Heidelberg
Greece Floor-raised 5/5 faeces, 0/40 swabs 0 % (0.00 – 7.18 %) S. Heidelberg
Italy Conv. Batt. 2/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Napoli, S. enterica subsp. ent.
Italy Conv. Batt. 3/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Virchow
Italy Conv. Batt. 4/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Enteritidis PT 27, NT
Italy Conv. Batt. 0/5 faeces, 2/40 swabs 5.00 % (0.00 – 11.75 %) S. Enteritidis, S. Bredeney
Italy Conv. Batt. 0/5 faeces, 1/40 swabs 2.50 % (0.00 – 7.34 %) S. Typhimurium NT
Italy Conv. Batt. 1/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. enterica subsp. enterica NT
Italy Floor-raised 3/5 faeces, 1/40 swabs 2.50 % (0.00 – 7.33 %) S. Bareilly, S. Thompson
Italy Free-range 2/5 faeces, 0/40 swabs 0 % (0.00 – 7.21 %) S. Kottbus
Italy Free-range 3/5 faeces, 1/40 swabs 2.50 % (0.00 – 7.33 %) S. Enteritidis PT 14, NT
Chapter 5 101
For the identification of risk factors only Salmonella Enteritidis and Typhimurium and
the following housing types were taken into account: conventional battery cages, indoor
production systems, free-range and free-range organic systems. The factors associated with
detection of Salmonella in the univariable analysis are listed in Table 5. In the final
multivariable logistic regression model (Table 5) the absence of dry cleaning (P < 0.01), the
housing type (P < 0.01) and the season of sampling (P = 0.01) turned out to be risk factors for
the shedding of Salmonella Enteritidis/Typhimurium in laying hen flocks.
Table 5: Results of the univariable and multivariate analysis for the identification of risk factors for
Salmonella Enteritidis or Typhimurium infection on 292 European laying hen farms.
Univariable analysis Multivariable analysis
Continuous variables
OR P-value OR 95 % CI
for OR P-value
Age of the infrastructure in years 1.07 < 0.01 / / /
Number of flocks in the sampled
house
1.39 0.04
/ / /
Number of egg collections per day 0.19 < 0.01 / / /
Categorical variable n OR P-value
Dry cleaning
No 52 13.49 < 0.01 14.37 4.54-45.51 < 0.01
Yes (ref) 240 - - - - -
Vaccinationstatus against Salmonella
No 210 2.62 0.13 / / /
Yes (ref) 82 - - / / /
Type of housing < 0.01 < 0.01
Conventional battery (ref) 59 - - - - -
Indoor production 11
1 0.09 < 0.01 0.05 0.01-0.24 < 0.01
Free-range 86 0.16 < 0.01 0.18 0.05-0.73 0.02
Organic 36 0.09 0.02 0.17 0.02-1.73 0.13
Season of sampling 0.02 0.01
Winter (ref) 49 - - - - -
Spring 80 0.30 0.04 0.16 0.04-0.73 0.02
Summer 97 0.09 < 0.01 0.06 0.01-0.40 < 0.01
Autumn 66 0.44 0.15 0.64 0.16-2.60 0.53
DISCUSSION
The housing of laying hens in conventional battery cages turned out to be a significant
risk factor for the shedding of Salmonella Enteritidis and/or Typhimurium. This is in
accordance with the results of the baseline study on the prevalence of Salmonella in laying
hen flocks, both at the EU level (EFSA 2007) and at the level of individual member states
(Methner et al., 2006; Namata et al., 2008; Huneau-Salaün et al., 2009). The reasons why a
102 Chapter 5
higher prevalence of Salmonella is found in cage housed laying hen flocks is likely to be a
combination of factors. First of all flock size may have an effect (Mollenhorst et al., 2005;
EFSA, 2007; Carrique-Mas et al., 2008; Huneau-Salaün et al., 2009). As also demonstrated in
this study, in cage farms flock sizes are in general larger in comparison to non-cage farms.
Second, the number of years that the current infrastructure is in use could also play an
important role. In this study the age of conventional battery cages was significantly higher
than that of the floor-raised, free-range and organic systems. In the univariable analysis the
age of the infrastructure was identified as a significant risk factor, as was the age of the
building. However, because there was a too strong correlation between these 2 variables, only
the first one was considered. The effect of the age of the infrastructure may be explained by
the fact that the older the infrastructure, the more difficult it gets to achieve sufficient
standards of cleaning due to the wear of the materials, both of the production system and of
the building itself, especially when it is taken into account that the level of environmental
contamination increases significantly during a production cycle (Wales et al., 2007). The
importance of such latent environmental sources of infections should not be underestimated
since Davies and Wray (1996) described the survival of Salmonella in empty poultry houses
for a period of 12 months. This implies the risk of „transfer‟ of an infection between
successive production cycles.
The multivariate model shows that “age of the infrastructure” does not remain
significant when combined with production type. This implies that this factor is not sufficient
to explain the risk conventional housing systems pose to Salmonella Enteritidis infections,
and thus that other factors related to the housing system are involved. One of these other
factors might be the fact that it is more difficult to thoroughly clean and disinfect cage
systems and therefore remainders from previous infections may be more difficult to eliminate
(Davies and Breslin, 2003; EFSA, 2007).
Another proof of the importance of cleaning is given by the fact that absence of dry
cleaning in between production rounds was found to be a significant risk factor for the
shedding of Salmonella. Dry cleaning is the mechanical removal of organic material (manure,
dust, feed spills…) before the wet cleaning (using water) is carried out. Only 82.2 % of the
sampled farms carried out dry cleaning of the laying hen houses where for wet cleaning and
disinfection this was 93.2 % and 92.5 % respectively. The value of dry cleaning in between
production rounds is 2-fold: on one hand it is very useful to remove organic matter that can
harbor Salmonella. On the other hand it contributes to a more efficient disinfection since the
Chapter 5 103
presence of considerable amounts of organic protective matter such as manure, dust and
spilled feed has an adverse effect on the efficacy of disinfection (Davies and Breslin, 2003;
Gradel et al., 2004).
In contrast to other studies (Bouwknegt et al., 2004; Mollenhorst et al., 2005; Namata
et al., 2008) but in accordance with Wales et al. (2007), a seasonable effect could be observed.
The odds to detect Salmonella were significantly higher in flocks that were sampled in winter
compared to flocks that were sampled in the other seasons of the year. This could be
explained by the fact that in housing systems with an outdoor run the hens are kept inside due
to wet and cold weather conditions. A high density of animals is a well-known risk factor for
Salmonella (EFSA, 2007; Huneau-Salaün et al., 2009). Furthermore, the air quality in laying
hen houses seems to be lower in winter (Ellen et al., 2000; Nimmermark et al., 2009). This
can cause stress in the hens, leading them from a Salmonella-carrying state to a Salmonella-
shedding state.
Because of reasons of multicollinearity, the protective influence of vaccination against
Salmonella could not be statistically confirmed. This finding will be further elaborated in the
general discussion section of this thesis.
The estimates of the within flock prevalence based on the cloacal swabs were usually
relatively low indicating that in general only a small percentage of birds in the positive flocks
were shedding the bacterium. It needs to be stressed that the estimates obtained are an
indication of the number of birds shedding Salmonella, and not necessarily an accurate
indication of the number of birds infected with Salmonella. It is likely that in a substantial
proportion of the birds sampled negative still some low level Salmonella infection may be
present. In that case the negative sample is due to the fact that the birds were not shedding the
bacteria at the moment of sampling or the used sampling technique was not sensitive enough
to detect the limited shedding. The sometimes large difference between the prevalence of
infected and shedding birds has recently been clearly demonstrated by Van Hoorebeke et al.
(2009). This also holds for the between flock prevalence.
Compared to the results of Belgium [27.7 % (22.1 – 33.9 %)], Germany [24.2 % (21.2
– 27.5 %)], Greece [25.7 % (20.5 – 31.6 %)] and Italy [7.9 % (5.9 – 10.5 %)] in the EFSA
baseline study on the between herd prevalence of Salmonella Enteritidis and/or Typhimurium
on laying hen holdings (EFSA, 2007), only in Belgium and Greece the prevalence detected in
our study, was lower. However, these findings should be interpreted with care because the
104 Chapter 5
farms that were contacted to be sampled were selected on the base of the housing type and
thus not random. In addition, the sampling design of this study is different from the one of the
EFSA baseline study.
CONCLUSION
The results of this study illustrate that, despite the fact that in non-cage housing
systems the chance of oro-faecal transmission of Salmonella is much higher than in
conventional battery cage systems, no higher prevalence of Salmonella could be observed in
flocks housed in these alternative systems. Several management and biosecurity measures
such as strict cleaning and disinfection practices have been identified as protective factors to
minimize the introduction and persistence of Salmonella on laying hen farms. In future, a
close follow up of the evolution in time, both of the prevalence of Salmonella spp. in laying
hen flocks housed in different housing systems and in the diversity of serovars isolated and
their significance for public health, will be necessary.
ACKNOWLEDGMENTS
This research was funded by the EU FP6, under the contract 035547 (Safehouse
project). The authors thank all farmers for their permission to sample their farm and all lab
technicians at the different institutions for their help during the bacteriological analyses.
Chapter 5 105
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EFSA, 2007. Report of the Task Force on Zoonoses Data Collection on the Analysis of the
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CHAPTER 6
THE EFFECT OF THE HOUSING SYSTEM ON
ANTIMICROBIAL RESISTANCE IN INDICATOR
BACTERIA IN LAYING HENS
S. Van Hoorebeke, F. Van Immerseel, A. C. Berge, D. Persoons, J. Schulz, J. Hartung, M.
Harisberger, G. Regula, L. Barco, A. Ricci, J. De Vylder, R. Ducatelle, F. Haesebrouck
and J.
Dewulf
Submitted in Epidemiology and Infection (2010)
110 Chapter 6
Chapter 6 111
ABSTRACT
The aim of this study was to determine the potential association between housing type and
multi-drug resistance in Escherichia coli and Enterococcus faecalis isolates recovered from
283 laying hen flocks. In each flock, a cloacal swab from four hens was collected and 1102 E.
coli and 792 E. faecalis isolates were recovered. Broth microdilution was used to test
susceptibility to antimicrobials including multiple drug resistance (MDR). A generalized
estimating equations logistic regression model was used to identify factors influencing MDR.
Country and housing type interacted differently with the MDR levels of both species. In the E.
coli model, the housing in a floor raised system was associated with an increased risk of MDR
in comparison to the conventional battery system. In the E. faecalis model, the MDR levels
were lower in free-range systems than in conventional battery cages. In Belgium, ceftiofur-
resistant E. coli isolates were more abundantly present than in the other countries.
Key words: antimicrobial resistance – E. coli – E. facalis – laying hen
112 Chapter 6
INTRODUCTION
The therapeutic, prophylactic and metaphylactic use of antimicrobials is common
practice in modern food animal husbandry (Berge et al., 2003; Aarestrup, 2004; EFSA, 2008).
Concerns have grown that this wide spread use of antibiotic drugs may lead to an increase in
antimicrobial resistance in numerous bacteria potentially affecting public health (EFSA, 2009;
Jordan et al., 2009). Standardized and continuous surveillance programs are necessary to
monitor the occurrence and persistence of antimicrobial resistance in food animals (Aarestrup,
2004; Wallmann, 2006; WHO, 2008). Indicator bacteria are generally used to monitor
antimicrobial resistance since they can be commonly found in healthy animals, giving an
indication of the level of resistance in a particular population. In addition, they acquire
antimicrobial resistance faster than other commonly found bacteria (Aarestrup, 2004; WHO,
2007; Miranda et al., 2008). Commensal Escherichia coli and Enterococcus faecalis are
internationally used as respective Gram-negative and Gram-positive indicator bacteriafor
monitoring antimicrobial resistance because of their common presence in the animal intestinal
tract (Wray and Gnanou, 2000; Sørum and Sunde, 2001; de Jong et al., 2009). Surveillance of
antimicrobial resistance is performed in several countries (Aarestrup, 2004), yet these
surveillance programs have generally been focused on cattle, pig and broiler production.
Programs in laying hens are still scarce. Therefore, there is a need to monitor antimicrobial
resistance development in laying hens (EFSA, 2008).
The Council Directive 1999/74/EC states that for welfare reasons as of January 2012
onwards conventional battery cages will be forbidden in the European Union (EU) (Wall et al.,
2004). Only enriched cages and non-cage housing systems will be allowed. Non-cage housing
systems consist of an indoor area that may or may not be combined with covered
(„wintergarden‟) or uncovered („free-range‟) outdoor facilities (EFSA, 2005; LayWel, 2006).
The non-cage systems can be categorized into 2 groups: single level systems with a ground
floor area which is fully or partially covered with litter and aviaries, consisting of a ground
floor area plus one or more platforms (EFSA, 2005; LayWel, 2006). Free-range organic
flocks have the same structure as a free-range system but there are some additional
requirements concerning maximum flock size, beak trimming and the origin of the feed.
Moreover, the application of antimicrobials in these organic flocks is strictly restricted to the
therapeutic usage. Presently it is not clear whether the different housing and management
systems for laying hens will influence the occurrence of antimicrobial resistance. Recent
reports indicate that both in poultry and other animal species the move from conventional
Chapter 6 113
indoor production towards free-range and organic production exerted a beneficial effect on
the levels of antimicrobial resistance in zoonotic and indicator bacteria (Avrain et al., 2003;
Thakur and Gebreyes, 2005; Ray et al., 2006, Schwaiger et al., 2008; 2010, Young et al.,
2009). On the other hand it has been shown that this move to non-cage housing systems
resulted in an increased incidence of particularly bacterial diseases (Fossum et al., 2009;
Kaufmann-Bart and Hoop, 2009), which could potentially lead to increased antibiotic usage.
However, epidemiological data on the prevalence of antimicrobial resistance in the above
mentioned indicator bacteria in laying hens in different housing systems is still limited. It is
therefore necessary to investigate the impact of these new laying hen housing systems on the
prevalence of antimicrobial resistance and to study antimicrobial resistance development in
laying hens (EFSA, 2008).
The aim of this study is to investigate the prevalence of antimicrobial resistance in E.
coli and E. faecalis isolates recovered from 283 laying flocks in four European countries and
to evaluate the potential association between housing systems and observed multi-drug
resistance.
MATERIAL AND METHODS
Farm selection
Flocks were selected from registry list of registered laying hen farms provided by the
official identification and registration authorities of the participating countries (Belgium,
Germany, Italy and Switzerland). The farm size (>1000 laying hens) and the housing type
were the only 2 selection criteria. The distribution of sampled farms aimed at was 20 %
conventional battery cage systems and 80 % (alternative) non-cage housing systems. Within
the group of alternative housing systems an equal number of aviaries, floor-raised, free-range
and free-range organic farms was targeted. The farmers were contacted by telephone and the
purpose of the study was explained. The foreseen date of depopulation was marked to make
sure that the farm could be sampled in the month prior to depopulation.
Sampling of the laying hen farms
The cloacal swabs were taken from randomly selected hens in each of the flocks as
carefully as possible to avoid unnecessary stress. Four hens in the flock were evenly selected
throughout the house and from each hen a cloacal swab was taken by inserting a sterile
cotton-tipped swab approximately 5 cm into the cloaca, taking care to avoid contact with the
114 Chapter 6
surrounding feathers and skin. The swabs were directly placed in tubes containing Ames
medium.
A questionnaire was completed during an on-farm interview at the same occasion as
collection of the samples took place. The questions were related to general farm and flock
characteristics such as flock size, breed, age of the hens and biosecurity measures. Special
attention was paid to the housing system of the sampled flock and the antimicrobial
treatments the hens had received during the current production cycle. The same questionnaire
was used in all participating countries. In each of the participating countries, there was one
person who collected the samples and did the interview with the farmer.
Bacteriological examination of the samples
For the isolation of E. coli the swabs were plated on MacConkey plates (Oxoid®,
France) and incubated aerobically for 24 h at 37°C. From each primary plate, one colony was
picked and plated again on a MacConkey agar plate. Suspected colonies were confirmed as E.
coli by positive glucose/lactose fermentation, gas production and absence of H2S production
on Kligler Iron Agar (Oxoid) and absence of aesculin hydrolysis (Bile aesculin agar; Oxoid).
Enterococcus was isolated on Slanetz & Bartley agar (Oxoid). After incubation for 48 h at 42
± 1°C, one suspected Enterococcus faecalis colony per sampled animal was purified and
verified by using Rapid ID 32 STREP strips (BioMérieux, France). In Switzerland, the
following equivalent methods were used: for the isolation of E. Coli, the cloacal swabs were
plated on MacConkey Agar (Oxoid) and incubated for 24 h at 37°C under aerobic conditions.
Strains which were lactose-positive were sub-cultured on Brolacin Agar (Merck®, Darmstadt,
Germany) and incubated for 24 h at 37°C under aerobic conditions. Confirmation of the
strains as E. coli was carried out using RapiD 20 E (BioMérieux). E. Faecalis were isolated
from the cloacal swabs on Enterococcosel Agar (Becton, Dickinson and Company, Franklin
Lakes, NJ, USA) and incubated for 48 h at 37°C under aerobic conditions. On Columbia 5%
Sheep Blood Agar (Oxoid) strains were sub-cultured and incubated for 24 h at 37°C under
anaerobic conditions. Confirmation of the strains as E. faecalis was carried out using Rapid
ID 32 STREP strips (BioMérieux).
Antimicrobial susceptibility testing
For both indicator bacteria, susceptibility was tested by means of broth microdilution
using custom made Sensititre® plates (Trek Diagnostics Systems Ltd., UK). The
antimicrobials tested and their ranges are listed in Tables 1 and 2 for E. coli and E. faecalis
Chapter 6 115
respectively. The results were read visually after 24 h of incubation at 37°C and the Minimum
Inhibitory Concentration (MIC) was defined as the lowest concentration of the antimicrobial
that completely inhibited visible growth. Escherichia coli ATCC 25922 and Enterococcus
faecalis ATCC 29212 were used for quality control. The EUCAST (European Committee on
Antimicrobial Susceptibility Testing) epidemiological breakpoints for MIC determination
were used, since epidemiological cut-off values are considered more useful than clinical break
points to achieve optimum sensitivity for detection of acquired resistance (Aarestrup et al.,
2007; EFSA, 2008). When these EUCAST breakpoints were not available (which was the
case for apramycin, sulfamethoxazole and kanamycin), the break points mentioned in the
reports of Danish Integrated Antimicrobial Resistance Monitoring and Research Program
were used (Danmap, 2007).
Table 1: Minimum Inhibitory Concentration distribution (%) for all Escherichia coli isolates (vertical black line indicates cut-off value)
Compound
Distribution (%) of MIC's (µg/ml) Total %
resistance 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1024 2048
ciprofloxacin 89.6 0.5 1.3 5.6 1.1 1.7 0.2 0.0 10.4
cefpodoxime 9.5 56.1 26.5 3.1 0.3 4.4 4.7
ceftiofur 94.6 0.8 0.4 0.4 3.8 4.6
ampicillin 5.2 32.0 43.2 3.6 0.0 15.8 15.8
gentamicin 94.5 3.0 0.9 0.3 0.7 0.6 2.5
amoxi/clav. acid 20.3 55.5 22.3 1.1 0.7 1.8
chloramphenicol 2.0 25.3 67.3 4.3 0.4 0.7 1.1
florfenicol 3.8 42.6 49.3 4.0 0.3 0.0 0.3
neomycin 90.6 3.4 0.5 0.4 5.1 5.5
tetracycline 73.8 2.9 0.5 0.0 22.8 22.8
colistin 0.0 0.9 0.0 0.9
streptomycin 66.9 20.5 3.4 1.8 6.9 8.7
apramycin 83.4 13.3 1.9 1.4 1.4
cefalothin 24.6 50.1 18.1 7.1 0.1
trimethoprim 90.2 0.5 0 8.9 9.4
nalidixic acid 87.4 1.5 0.4 0.4 10.3 10.7
spectinomycin 68.8 22.5 5.9 1.5 1.3 2.8
sulfamethoxazole 81.1 0.8 0.8 0.2 17.0 17.2
Chapter 6 117
Statistical data analysis
The Pearson‟s χ2-test was used to determine significant differences between
categorical variables (P < 0.05), such as the presence of other animal production on the farm
and the treatment status of the flock. An isolate was defined as multiple drug resistant (MDR)
if it exhibited resistance to 2 or more antimicrobials. For both indicator bacteria, MDR was
used as an outcome variable in the statistical analysis. For E. coli, ceftiofur-resistance was
also used as an outcome variable. The exploratory factors, consisting of country, housing type,
presence of other farm animals (pigs, cattle, sheep…) on farm, the presence of hens in the
flock originating from different rearing sites and antimicrobial treatment of the flock were
tested for inclusion in the models. To identify influential factors a stepwise forward selection
process was used for the variable selection in a population average logistic regression model
with a p-value ≤ 0.2 for entry, and with a p-value ≤ 0.10 for retention in model. The factors
that were significant in this model were introduced into a model using generalized estimating
equations (GEE) to control for clustering of samples within a farm using an independent
correlation matrix. Interaction effects were tested for variables retained in the GEE model.
Odds ratios and 95% confidence intervals were calculated for the parameters that were
retained in the GEE model using the criteria of p-value ≤ 0.05 for retention in the GEE model.
The statistical software package SAS (SAS for Windows, version 9.1, SAS Institute Inc, Cary,
NC, USA) was used for data analysis.
Antimicrobial resistance patterns were described using cluster analysis. Binary cluster
analysis was performed using the Jaccard matching coefficient and the centroid method to
obtain discrete clusters with no intra-cluster variability. Due to the large number of
antimicrobial resistance clusters, the descriptive and stratified analysis was limited to the 15
most common resistance patterns describing more than 80 % of the isolate data set.
Table 2: Minimum Inhibitory Concentration distribution (%) for all Enterococcus faecalis isolates (vertical black line indicates cut-off value)
Compound
Distribution (%) of MIC's (µg/ml) Total %
resistance 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1024 2048
tigecycline 0.1 0.1 25.8 45.4 2.1 4.1 21.8 0.2 26.1
daptomycin 1.5 0.4 3.2 42.8 42.6 7.3 0.8 0.3 1.1
ciprofloxacin 7.7 20.5 59.8 7.3 4.6 0.1 0.1
erythromycin 19.7 19.5 19.6 6.3 2.6 1.6 31.3 35.5
tetracycline 30.4 0.4 0.6 0.1 1.5 67.0 68.6
linezolid 18.2 79.2 2.2 0.4 0.4
chloramphenicol 2.1 29.0 65.8 2.6 0.3 0.3 0.3
vancomycin 96.4 3.1 0.2 0.1 0.1 0.4
ampicillin 93.5 5.2 0.7 0 0.6 0 1.3
avilamycin 94.4 3.8 0.6 1.1 1.1
salinomycin 98.6 1.2 0.1 0.1 0.2
florfenicol 98.0 1.6 0.1 0.2 0.3
gentamicin 97.7 0.4 0.1 0 1.8 1.8
kanamycin 77.1 4.3 1.2 0.8 16.4 16.4
streptomycin 74.0 1.6 0.7 0.8 22.7 23.5
Chapter 6 119
RESULTS
In total, 1102 E. coli isolates and 792 E. faecalis isolates were collected from 283
laying hen flocks (69 Belgian, 85 German, 30 Italian and 99 Swiss flocks). The participation
rate was more than 90 % in Belgium, Italy and Switzerland and 70 % in Germany. A detailed
description of the number of isolates per housing type and per country is presented in Table 3.
Twenty-four of the 283 sampled flocks were treated with antimicrobials according to
the declaration of the farmers. The number of treated flocks differed significantly between
countries (P < 0.05) but not between housing types. In Italy none of the sampled flocks were
treated with antimicrobials, for Belgium this was 3 of the 69 sampled flocks, for Switzerland
5 of the 99 flocks and for Germany 16 of the 85 sampled flocks. Colistin was the most
frequent treatment (16 flocks), followed by amoxicillin (5 flocks), neomycin (2 flocks) and
enrofloxacin (1 flock). The antimicrobial treatments per flock and per housing type are
presented in Table 4. There was a significant difference between countries in the number of
laying hen farms where also other production animals were kept (P < 0.05). In Italy, only
16.7 % of the sampled laying hen farms managed other animal production on the same site,
whereas in Germany, Belgium and Switzerland this was 38.5 %, 43.8 % and 75.5 %
respectively.
Table 3: Number of isolates per country and per housing type for E. coli and E. faecalis Belgium Germany Italy Switzerland
No. of
flocks
No. of
isolates
No. of
flocks
No. of
isolates
No. of
flocks
No. of
isolates
No. of
flocks
No. of
isolates
Battery E. coli 18 72 26 101 12 47 - -
E. faecalis 18 68 26 102 12 41 - -
Aviary E. coli 1 4 - - - - 36 138
E. faecalis 1 4 - - - - 36 42
Floor-raised E. coli 21 79 21 98 10 40 16 64
E. faecalis 21 64 21 92 10 31 16 17
Free-range E. coli 21 69 24 92 8 32 33 116
E. faecalis 21 68 24 71 8 23 33 49
Free-range organic E. coli 8 32 14 65 - - 14 53
E. faecalis 8 32 14 58 - - 14 30
Total E. coli 69 256 85 356 30 119 99 371
E. faecalis 69 236 85 323 30 95 99 138
Chapter 6 121
The distribution of the MIC‟s (in %) of each antimicrobial is described in Table 1 for
E. coli and Table 2 for E. faecalis. The isolates were grouped according to their multi-
resistance patterns, resulting in 120 and 94 antimicrobial resistance (AR)-clusters for E. coli
and E. faecalis respectively. Due to space limitations, only the top 15 clusters for each
bacterial species are shown (complete descriptions of patterns can be obtained from
corresponding author upon request).
Escherichia coli
The majority of the isolates (55.0 %) were susceptible to all 18 antimicrobials, 16.9 %
were resistant to 1 antimicrobial and the remaining 28.1 % were multi-resistant. Only to
tetracycline (22.8 %), sulfamethoxazole (17.2 %), ampicillin (15.8 %), ciprofloxacin (12.3 %)
and nalidixic acid (10.7 %), more than 10 % of all isolates showed resistance. The 15 most
common resistance phenotypes of AR-clusters are described in Table 5. These clusters
included 81.8 % of the isolates.
Table 4: Antimicrobial treatments in 292 European laying hen flocks
Country Housing system Antimicrobial agent
Belgium Conventional battery cage colistin
Belgium Floor-raised colistin
Belgium Free-range colistin
Germany Conventional battery cage colistin
Germany Conventional battery cage colistin
Germany Conventional battery cage colistin
Germany Conventional battery cage colistin
Germany Floor-raised colistin
Germany Floor-raised colistin
Germany Floor-raised colistin
Germany Floor-raised colistin
Germany Free-range colistin
Germany Free-range colistin
Germany Free-range colistin
Germany Free-range colistin
Germany Free-range colistin
Germany Conventional battery cage amoxicillin
Germany Free-range neomycin
Germany Free-range neomycin
Switzerland Aviary amoxicillin
Switzerland Aviary amoxicillin
Switzerland Aviary amoxicillin
Switzerland Aviary amoxicillin
Switzerland Floor-raised enrofloxacin
Table 5: The 15 most common antimicrobial resistance clusters in E. coli isolated from European laying hens
Antimicrobial agents
AR-
cluster1
No. of
isolates
%
AR-
Freq2
AMC
AMP
APR
CEF
CEP
CHL
CIP
COL
FLO
GEN
NAL
NEO
SPT
STR
SUL
TET
TRI
XNL
1
606
55.0
0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
2 58 5.3 1 . . . . . . . . . . . . . . . R . .
3 47 4.3 1 . R . . . . . . . . . . . . . . . .
4 30 2.7 2 . . . . . . R . . . R . . . . . . .
5 26 2.4 1 . . . . . . . . . . . R . . . . . .
6 25 2.3 1 . . . . . . . . . . . . . . R . . .
7 23 2.1 3 . . . . . . R . . . R . . . . R . .
8 16 1.5 3 . . . . . . R . . . R . . . R . . .
9 15 1.4 2 . R . . . . . . . . . . . . . R . .
10 14 1.3 3 . . . . . . . . . . . . . . R R R .
11 9 0.8 3 . R . . . . . . . . . . . . R . R .
12 9 0.8 6 . R . R R . . . . . . . . . R R . R
13 8 0.7 1 . . . . . . . . . R . . . . . . . .
14 7 0.6 6 . R . . . . R . . . R . . R R R . .
15 7 0.6 9 . R . R R . . . . . . R . R R R R R
Sum3
900
81.8
AMC: amoxicillin/clavulanic acid, AMP: ampicillin, APR: apramycin, CEF: cefpodoxime; CEP: cefalothin, CHL: chloramphenicol; CIP: ciprofloxacin, COL: colistin, FLO: florfenicol, GEN:
gentamicin, NAL: nalidixic acid, NEO: neomycin, SPT: spectinomycin, STR: streptomycin, SUL: sulfamethoxzole, TET: tetracycline, TRI: trimethoprim, XNL: ceftiofur
1AR-cluster = Antimicrobial Resistance cluster describing the pattern of resistance of the isolates
2AR-Freq = Antimicrobial Resistance frequency: the number of antimicrobials to which the E. coli were classified as resistant (R)
3The number and percentage of isolates described in this table, representing 81.8 % of the 1102 total isolates included in this study
Chapter 6 123
The housing of hens in floor-raised systems, compared to conventional battery cages
(P = 0.02) and the country (P = 0.03) turned out to be risk factors for higher levels of MDR in
the final GEE logistic regression model (Table 6). Other factors such as other animal
production on the farm, the presence of hens originating from different rearing farms in the
sampled flock and antimicrobial treatment of the flock were not retained in the final model.
Table 6: Results of the GEE logistic regression analysis for the identification of risk factors for the
presence of multiple drug resistance in Escherichia coli from European laying hens.
Multivariable analysis
Categorical variable N of isolates OR 95 % CI P-value
Type of housing
Conventional battery (ref) 217 - - -
Aviary 142 0.87 0.90-2.26 0.77
Floor-raised 284 2.12 1.13-3.97 0.02
Free-range 309 0.84 0.42-1.68 0.62
Free-range organic 150 1.02 0.45-2.33 0.96
Country
Belgium (ref) 256 - - -
Germany 356 0.54 0.31-0.93 0.03
Italy 119 0.73 0.38-1.42 0.36
Switzerland 371 0.86 0.48-1.60 0.67
When looking at factors affecting ceftiofur resistance in E. coli (Table 7), Belgium
was more likely to have ceftiofur-resistant isolates than the three other countries. Ceftiofur-
resistance varied from 0.0 % (Switzerland), over 2.5 and 4.5 % (Italy and Germany), to 12.1 %
in Belgium. No significant potential association between the other tested risk factors (housing
system, other animal production on the same farm and hens originating from different rearing
farms) was observed.
Table 7: Results of the GEE logistic regression analysis for the identification of risk factors for the
presence of ceftiofur resistance in Escherichia coli from European laying hens.
Multivariable analysis
Categorical variable N of isolates OR 95 % CI P-value
Country
Belgium (ref) 256 - - -
Germany 356 0.72 0.42-1.24 0.24
Italy 119 0.62 0.29-1.32 0.21
Switzerland 371 0.40 0.21-0.76 <0.01
124 Chapter 6
Enterococcus faecalis
The majority of E. faecalis isolates were multiresistant (51.1 %), 34.5 % were resistant
to 1 antimicrobial and only 14.4 % of all isolates were pan-susceptible. Resistance to
tetracycline (68.6 %) was most common, followed by erythromycin (35.5 %), tigecyclin
(26.1 %), streptomycin (23.5 %) and kanamycin (16.4 %). The 15 most common resistance
phenotypes of AR-clusters representing 81.2 % of the dataset are described in Table 9.
The results of the GEE logistic regression model, showing factors associated with
MDR in E. faecalis are presented in Table 8. The isolates from laying hens housed in free-
range systems were more likely to have lower levels of MDR (P = 0.03) compared to
conventional battery cage systems. Isolates from Belgian hens had lower levels of resistance
than hens in Germany and Italy. Similar to the observations in E. coli, other factors such as
other animal production on the farm, antimicrobial treatment of the flock and the presence of
hens originating from different rearing plants in the flock did not significantly interact with
the levels of MDR.
Table 8: Results of the GEE logistic regression analysis for the identification of risk factors for the
presence of multiple drug resistance in Enterococcus faecalis from European laying hens.
Multivariable analysis
Categorical variable N of isolates OR 95 % CI P-value
Type of housing
Conventional battery
(ref)
211 - - -
Aviary 50 1.13 0.39-3.28 0.83
Floor-raised 207 0.95 0.52-1.72 0.85
Free-range 214 0.51 0.27-0.94 0.03
Free-range organic 121 0.57 0.28-1.18 0.13
Country
Belgium (ref) 236 - - -
Germany 323 2.67 1.57-4.55 <0.01
Italy 95 13.07 5.69-30.00 <0.01
Switzerland 149 1.86 0.91-3.79 0.09
Table 9: The 15 most common antimicrobial resistance clusters in E. faecalis isolated from European laying hens
Antimicrobial agents
AR-
cluster1
No. of
isolates
%
AR-
Freq2
AMP
AVI
CHL
CIP
DAP
ERY
FLO
GEN
KAN
LIN
SAL
STR
TET
TIG
VAN
1
138
15.1
1
.
.
.
.
.
.
.
.
.
.
.
.
R
.
.
2 116 14.4 0 . . . . . . . . . . . . . . .
3 89 11.1 1 . . . . R . . . . . . . . . .
4 70 8.7 2 . . . . R . . . . . . . R . .
5 48 6.0 4 . . . . . R . . R . . R R . .
6 42 5.2 5 . . . . R R . . R . . R R . .
7 41 5.1 2 . . . . . R . . . . . . R . .
8 28 3.5 2 . . . . . . . . . . . . R R .
9 23 2.9 4 . . . . R R . . . . . R R . .
10 23 2.9 3 . . . . R R . . . . . . R . .
11 13 2.1 4 . . . . R R . . R . . . R . .
12 13 1.6 3 . . . . . R . . . . . R R . .
13 7 0.9 3 . . . . R . . . . . . R R . .
14 7 0.9 2 . . . R R . . . . . . . . . .
15 6 0.8 4 . . . . . R . . . . . R R R .
Sum3
664
81.2
AMP: ampicillin, AVI: avilamycin, CHL: chloramphenicol; CIP: ciprofloxacin, DAP: daptomycin, ERY: erythromycin, FLO: florfenicol, GEN: gentamicin, KAN: kanamycin, LIN: linezolid,
SAL: salinomycin, STR: streptomycin, TET: tetracycline, TIG: tigecyclin, VAN: vancomycin
1AR-cluster = Antimicrobial Resistance cluster describing the pattern of resistance of the isolates
2AR-Freq = Antimicrobial Resistance frequency: the number of antimicrobials to which the E. faecalis were classified as resistant (R)
3The number and percentage of isolates described in this table, representing 81.2 % of the 803 total isolates included in this study
126 Chapter 6
DISCUSSION
In this study, the alternative non-cage production systems for laying hens did not show
consistent results with regard to MDR in E. coli and E. faecalis. The levels of MDR in E.
faecalis were lower in free-range laying hens than in the conventional battery cage system,
whereas increased levels of MDR were seen in E. coli in floor-raised hens. This is different
from the studies of Schwaiger et al. (2008; 2010) who found significantly lower levels of
antimicrobial resistance in E. coli and faecal enterococci in organic laying hens compared to
laying hens housed in conventional battery cages.
There are several possible explanations for the ambiguous association between the
non-cage housing types on the level of MDR. A first important factor is the exposure to
antimicrobials. In the current study, apart from the free-range organic flocks, the reported
antimicrobial use in laying hens in the alternative systems was not significantly lower than in
conventional battery cages. Maybe the higher incidence of bacterial diseases in laying hens
housed in alternative non-cage systems that has been described in several studies (Fossum et
al., 2009; Kaufmann-Bart and Hoop, 2009) may have resulted in a more frequent use of
antimicrobials. If this would be the case, it raises the question whether, besides the advantages
on the animal-welfare level, in the future there won‟t be any adverse consequences for public
health on the level of the spread and persistence of antimicrobial resistance in laying hens.
Secondly, in non-cage systems the chance of oro-faecal transmission of bacteria is much
higher than in conventional battery cages, both between hens and between the animals and the
environment. This could also be of importance since, apart from antimicrobial usage, other
factors such as the localization and size of the microbial population (van den Bogaard and
Stobberingh, 1999), immunity and contact intensity of the host (Walson et al., 2001) play a
role in antimicrobial resistance development. Finally, the fact that many of the sampled farms
with non-cage production systems made the move to the new production systems only a few
years before the onset of the study and that these new production systems are often located in
the same house where previously the battery cages were present, might also explain the
observed results. It has been described that the decrease in antimicrobial resistance after
changes in the production system or in the antimicrobial usage policy in food producing
animals is very slow and that resistance against certain antimicrobials can be detected even
long after direct selection pressure by using antimicrobials came to an end (Dunlop et al.,
1998; De Gelder et al., 2004).
Chapter 6 127
The difference in effect of the housing system on MDR in E. coli and E. faecalis
might be the result of different biological characteristics of both bacterial species. E. coli is
typical an inhabitant of the intestinal tract and is to a considerable extent present in the hens‟
faeces (Huang et al., 2009). The animals have frequent oro-faecal contacts in non-cage indoor
production systems, resulting in an intense exchange of E. coli between hens. This high
contact intensity could cause the higher levels of MDR in laying hens in floor-raised systems.
Since enterococci are widely distributed in the soil and the environment (Aarestrup, 2002), the
access to a pasture may exert some kind of diluting effect on the intestinal enterococcal
population, leading to lower levels of MDR in free-range laying hens. However, although
these differences between the housing types are statistically significant, it is not yet clear
whether they have some biological relevance and therefore further study is necessary to
confirm and clarify these findings.
Although differences in methodology of sampling and analysis between different
studies have to be taken into account, the results of this study illustrate that, in general, the
levels of antimicrobial resistance in indicator bacteria in laying hens are relatively low
compared to broilers, pigs and –to a lesser extent− cattle (Bywater et al., 2004; de Jong et al.,
2009; Persoons et al., 2009). This is in accordance with previous studies in laying hens
(Schwaiger et al., 2008; 2010; Kojima et al., 2009). The overall limited antimicrobial usage in
egg-producing laying hens, as shown in the results, could play a role in this observation since
it is generally accepted that the use of antimicrobials is one of the major risk factors for the
development and spread of antimicrobial resistance (Wegener et al., 1999; van den Bogaard et
al., 2001; Johnsen et al., 2009). A reason for the lower levels of resistance seen in the layers
compared to broilers may be that the layers in this study were sampled on average at 74 weeks
of age, compared to broilers that are usually 6 weeks at the time of slaughter. It has been
described indeed for several animal species that antimicrobial resistance levels decrease with
increasing age (Langlois et al., 1988; Butaye et al., 1998). From this point of view, it would
be very interesting to carry out longitudinal studies to see the interaction between
antimicrobial resistance and the age of the hens and to monitor the use of antimicrobials
during the rearing of the pullets.
The fact that the samples were analyzed in different labs in different countries may
have slightly influenced the results, despite the equivalent methodology used in all 4
participating countries. However, the observed differences in MDR levels between countries,
probably also result from regional differences in animal husbandry and antimicrobial usage
128 Chapter 6
and from the fact that the distribution of the housing system of the sampled farms was not the
same in each country. A remarkable finding in this respect is the ceftiofur resistance in E. coli
in Belgium. Whereas in Germany, Italy and Switzerland only very low levels of ceftiofur
resistance were found, in Belgium this resistance was 12.1 % in E. coli. Consequentially the
odds to have resistance against ceftiofur were higher in Belgium compared to the other
countries, although only the difference with Switzerland was significant. This study result
coincides with recent findings of high levels of ceftiofur resistance in broilers in Belgium
(Persoons et al., 2009). It has recently been stated by several authors that one of the reasons
for the increasing levels of ceftiofur resistance may be the worldwide and systematic use of
ceftiofur in breeding eggs and one-day-old chicks in the hatcheries (Collignon and Aarestrup,
2007; CMAJ, 2009; Webster, 2009). In Belgium the use of ceftiofur in poultry has not been
licensed for a decade (Bertrand et al., 2006) but might be continued off-label (Persoons et al.,
2009). This is an additional argument to include laying hens in antimicrobial resistance
monitoring programs. Also monitoring of the use of antimicrobials during the rearing of the
pullets needs attention since it has been described for pigs and poultry that the younger
animals exhibit a higher prevalence of resistance than the full-grown animals (Langlois et al.,
1988; Butaye et al., 1998). To our knowledge, data on antimicrobial use and antimicrobial
resistance in pullets are extremely scarce.
Another hypothesis for the increased ceftiofur resistance might be the production on
the same farm of other animal species, in which the use of ceftiofur is still registered. This
could result in horizontal transmission of resistance genes between bacterial populations of
different animal species (Kruse and Sorum, 1994; Lorenz and Wackernaegel, 1994). However,
the number of mixed-production farms in Belgium did not significantly differ from the
situation in Germany and was even less than in Switzerland. Possibly the high density of
farms in the northern part of Belgium, where 90 % of all animal production is situated, may
enhance contact between the bacterial populations of different ecological systems, for
example surface water, leading to an efficient horizontal spread of antimicrobial resistance.
For humans, it has been demonstrated that a higher population density enhances the
development and spread of antimicrobial resistance (Garau et al., 1999; Bruinsma et al., 2003).
Further eco-epidemiological studies are needed to elucidate this possibility.
Chapter 6 129
CONCLUSION
The results of this study suggest that the levels of antimicrobial resistance in indicator
bacteria such as E. coli and E. faecalis in laying hen flocks are relatively low. The differences
observed between both indicator bacteria with respect to the potential association between the
housing system and MDR suggest that it is important to not focus on a sole bacterial species
when trying to assess risk factors for antimicrobial resistance. It is crucial to conscientiously
monitor the prevalence and evolution in time of antimicrobial resistance in laying hens, both
during the rearing period and the production cycle, to be able to detect early changes in
antimicrobial resistance and to minimize the spread of resistant bacteria to humans. Therefore
it is recommended to carry out detailed epidemiological studies in the field and under
experimental conditions on a broader spectrum of indicator bacteria.
ACKNOWLEDGEMENTS
This research was funded by the EU FP6, under the contract 035547 (Safehouse
project). The authors thank all farmers for their permission to sample their farm and all lab
technicians at the different institutions for their skillful help during the bacteriological
analyses.
130 Chapter 6
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CHAPTER 7
GENERAL DISCUSSION
Chapter 7 137
The scope of this thesis was to determine if, and to what extent, the move from battery
cages to non-cage housing systems influences the prevalence of Salmonella and antimicrobial
resistance in laying hens and to identify risk factors for the presence of Salmonella in laying
hen flocks in different housing systems.
1. SAMPLING OF LAYING HEN FLOCKS: DETECTING THE SHEDDING OR THE
CARRIAGE OF SALMONELLA?
It has been described that a vaccinated flock is not guaranteed Salmonella-free, a
finding which is corroborated by the results presented in chapter 3. In this study, 19
vaccinated laying hen flocks were extensively sampled at the end of the production cycle,
both on-farm (5 pooled faeces samples, 40 cloacal swabs and 1 mixed dust sample) and after
transport (cloacal swabs of 100 hens, caeca of 100 hens). All of the sampled farms were found
negative for Salmonella according to the official monitoring program.
The data of chapter 3 suggest that on farms which are „apparently Salmonella-free‟, a
relatively large proportion of the flocks may still carry the pathogen without shedding.
Therefore, the numbers of infected flocks based on the official monitoring programs are most
likely an underestimation of the true number of flocks in which Salmonella is still present.
The actual public health risk presented by these positive flocks is very difficult to
assess. The fact that Salmonella is still present in apparently negative flocks implies the risk
of a renewed shedding of the pathogen, especially at moments of stress, leading to egg
contamination. Shedding of the pathogen can cause surface contamination of the eggs,
following oviposition (Gantois et al., 2009). Moreover, the pathogen can easily penetrate
through cracked egg shells, leading to internal egg contamination (Braden, 2006).
However, based on the observation that using the conventional sampling methodology
no positive samples were found (no indication of active shedding) and only a limited number
of shedders and carriers were found after submitting the birds to transport stress and intensive
sampling, one can argue that the risk of egg contamination in these vaccinated flocks is
probably very low because of the low infection pressure. It has been described that the lower
the degree of environmental contamination, the lower the numbers of contaminated eggs
produced by an infected flock (Henzler et al., 1998; Chemaly et al., 2009).
Moreover, these results indicate that the sampling methodology as it is currently used
in the Salmonella monitoring programs of several EU member states might not be accurate
138 Chapter 7
enough to detect Salmonella in flocks where the bacterium is only shed in very small amounts,
or not shed at all. It can be expected that in the forthcoming years these low-level Salmonella
infections will more regularly occur because of measures such as mandatory vaccination and a
more thorough sensibilization of the farmers.
As described in the general introduction of this thesis, the current monitoring
programs are based on the collection of pooled faeces samples or overshoes. However, even
the intensified on-farm sampling, combining bacteriological analysis of 40 cloaca swabs, 1
mixed dust sample and 5 pooled faeces samples, did not result in the detection of Salmonella.
The absence of Salmonella in pooled faeces and cloacal swabs in these flocks can be
attributed to several factors. The first one is the very low degree of shedding at the moment of
sampling. This can be the result of the vaccination against Salmonella or it can simply be the
result of the intermittent excretion of Salmonella by infected animals (Van Immerseel et al.,
2004). Another factor possibly interfering with the detection of Salmonella in faeces is the
acidification of the drinking water. A wide variety of products for Salmonella control are
available, claiming a decrease in shedding of the bacterium in the treated laying hens (Van
Immerseel et al., 2002). At the same time, it raises the question whether the inhibited
shedding caused by the use of these additives does not lead to failure to detect infected flocks.
In the study described in chapter 4, 75 % of the sampled flocks and 5 out of 6 positive flocks
were supplied with acids in the drinking water. All this can also be an explanation why the
individual cloacal swabs taken on-farm did not seem to be appropriate to detect Salmonella in
the flocks, which is in accordance with the results of Bichler et al. (1996) and Van Immerseel
et al. (2004). Analyzing cloacal swabs of laying hens can give an indication of the number of
shedding birds but it is not indicative of the number of hens carrying the pathogen.
The mixed dust sampling yielded no positive results although the collection and
analysis has often been described as a suitable sample type to detect Salmonella in a laying
hen house (Davies and Wray, 1996; Kwon et al., 2000; Davies and Breslin, 2003; Gast et al.,
2004). Again, the low level of excretion in the sampled flocks could have played a role.
Arnold et al. (2009) demonstrated that the sensitivity of dust samples increases when the
within-flock prevalence of shedding birds, and thus the infection pressure, increases. Other
factors that are thought to influence the efficiency of dust sampling are the stage of infection
and the housing type.
The results of this thesis indicate that the post-mortem examination of hens (caecal
and/or ovary/oviduct samples) is the most accurate test to determine the infection status of a
Chapter 7 139
laying hen flock. This way of sampling also allows estimating the number of infected hens
within a flock. However, the fact that it is an expensive and time-consuming technique makes
it impossible to use it in routine monitoring programs. To circumvent this, a possible solution
could be the post-mortem examination of culled hens in the slaughterhouse. In this way, data
on the between- and within-flock prevalence could be obtained without any healthy hen killed
during the production round. The sampling of culled hens in the slaughterhouse also has some
disadvantages. First there is the risk of cross-contamination, not only between hens of the
same flock but also between hens of different flocks or farms, both during transport and
processing. This would lead to an over-estimation of the prevalence of Salmonella. Therefore,
strict cleaning and disinfection measures are necessary. Also the risk of infection of laying
hens during transport needs to be taken into account. Another major drawback of sampling
culled hens is that the information on the Salmonella status of the flock is only obtained after
the eggs are sold or processed. In this way, the information forming the base on which the
public health risk presented by such flock is assessed, comes too late. This is one of the main
differences between the control of Salmonella in laying hens and other food producing
animals: in broilers and pigs the level of Salmonella contamination can be heavily influenced
by the different processing steps (e.g. transport to the slaughterhouse, hygiene along the
slaughter line, storage after processing…), whereas in the egg industry the prevalence of
Salmonella contaminated eggs changes only very little between the time the eggs are laid and
the consumption of the eggs. Therefore, Salmonella control in laying hens should be focused
to an even larger extent at the level of the primary production, i.e. the laying hen farms.
In this respect, the combination of the on-farm sampling as it is currently performed
and the testing of culled hens in the slaughterhouse would give the most complete information
on the infection status of a flock. The shedding of Salmonella could be monitored using the
on-farm sampling, the presence of hens carrying Salmonella could be detected in the
slaughterhouse. Any positive results in the caecal or ovarian samples would imply that extra
measures, for instance cleaning and disinfection in accordance with official guidebooks, must
be taken before the house can be restocked with new pullets.
2. PREVALENCE OF SALMONELLA IN EUROPEAN LAYING HEN FLOCKS
In the cross-sectional field study carried out during this thesis (chapter 5), 29 of the
292 sampled flocks were found positive for Salmonella in at least one of the samples taken.
The between flock prevalences differed significantly between the countries, ranging from 0.0 %
140 Chapter 7
(Switzerland) to 30.0 % (Italy). The prevalence in each country with the 95 % CI is presented
in Table 1. Salmonella Enteritidis is still the most commonly isolated serovar: in 21 of these
29 positive flocks S. Enteritidis could be found.
Table 1: Prevalence of Salmonella in 292 European laying hen flocks
Salmonella spp. Salmonella Enteritidis
and/or Typhimurium
Country
No. of flocks
% pos
CI 95 %
% pos
CI 95%
Belgium 69 1.4 0.0-3.7 1.4 0.0-3.7
Germany 84 20.0 11.7-28.7 20.0 11.7-28.7
Greece 10 20.0 0.0-44.6 0.0 0.0-2.4
Italy 30 30.0 13.8-46.2 13.3 1.3-25.3
Switzerland 99 0.0 0.0-3.7 0.0 0.0-3.7
Total 292 9.9 6.7-13.1 7.5 4.7-10.3
When comparing these results to the results of the EFSA baseline study, only in
Belgium and Greece a significantly lower prevalence of S. Enteritidis and/or Typhimurium
could be observed. For several reasons these findings should be interpreted with care. First of
all, the farms in this study were not randomly sampled but they were selected based on the
housing type. This will certainly have influenced the outcome of the study. The fact that
conventional battery cage farms only accounted for 25 % of the sampled farms most probably
lead to an under-estimation of the prevalence. The influence of the housing system on the
presence of Salmonella in laying hens will be discussed further on in the section about the risk
factors. Secondly, participation of the contacted farmers in this study was voluntarily, which
could have caused bias. The participation rates for Belgium, Greece, Italy and Switzerland
were > 90 % since absolute anonymity could be guaranteed to the participating farmers;
therefore a positive result could not have any adverse consequences for the farmer. For
Germany the participation rate was lower (70 %) since positive results had to be reported to
the authorities. Furthermore the German farmers were awaiting the decision on the housing of
laying hens in the so called German type of enriched cages. This uncertainty caused some
reluctance to participate in this particular study. On the other hand, the fact that also red mites
were collected for further research was a big stimulus for farmers to participate since these
ectoparasites are of major concern for the farmers and are perceived much more as a problem
than Salmonella is. Finally, the sampling scheme used in this study was different from the one
of the EFSA baseline study. In our study 5 pooled faeces samples and 40 cloacal swabs were
Chapter 7 141
collected from each flock. These samples are suitable for detecting the excretion of
Salmonella but not for detecting carriers of Salmonella. In the EFSA baseline study 5 pooled
faeces samples and 2 mixed dust samples were collected. It is thought that the sampling of
dust permits the detection of Salmonella even when no hens are shedding at the moment of
sampling: the bacterium‟s capacity to resist long-term desiccation (Haysom and Sharp, 2003)
makes that settled dust in laying hen houses can give more information on the past Salmonella
status of the poultry house (Arnold et al., 2009).
For Belgium, it is remarkable that the prevalence of S. Enteritidis and/or Typhimurium
was much lower in this study compared to the EFSA baseline study (1.43 % vs. 26.2 %). It
has been stated that the large-scale vaccination of commercial laying hens since 2005,
together with sanitary and prevention campaigns, has been of major importance for the
decline of S. Enteritidis (Collard et al., 2008).
From this point of view, it may seem strange that vaccination against Salmonella did
not turn out to be a significant protective factor when all 292 flocks sampled in the cross-
sectional study were taken into account. After all, the commonly used vaccines claim to
reduce the faecal shedding and systemic spread of S. Enteritidis and to reduce the
reproductive tract colonization (Van Immerseel et al., 2004; Gantois et al., 2006). The fact
that the protective effect of vaccination could not be confirmed in our study is due to the
different vaccination policies and Salmonella prevalences in the participating countries.
Therefore vaccination policy and country were strongly correlated and resulted in
multicollinearity. Although in Belgium the vaccination of commercial laying hens became
mandatory only in 2007, vaccination already occurred in large scale since 2005 (Collard et al.,
2008). In Switzerland on the other hand, vaccination of laying hens against Salmonella is
prohibited. In Germany, Greece and Italy vaccination occurred on a voluntary basis,
explaining why in these three countries respectively 1.2 %, 0 % and 40 % of the sampled
flocks were vaccinated. Together with the large national differences in S. Enteritidis and/or
Typhimurium prevalence, no well-founded conclusion on the protective effect of vaccination
could be drawn on the basis of the results of our cross-sectional study.
142 Chapter 7
3. RISK FACTORS FOR SALMONELLA INFECTION: INFLUENCE OF THE AGE OF THE
PRODUCTION SYSTEM AND PREVIOUS SALMONELLA SHEDDING
One of the aims of this thesis was to determine risk factors for an infection with
Salmonella in vaccinated laying hen flocks, meaning that factors were identified that could
explain why in some flocks the risk was higher to find Salmonella, even when there were no
birds shedding the bacterium. The 2 variables which turned out to be significant were a) the
age of the production system and b) previous Salmonella shedding on the farm. The housing
system as such was not significantly associated with Salmonella infection.
Age of the infrastructure
For conventional battery farms the age of the infrastructure is the number of years the
current cages are in use, for non-cage systems this is the number of years the current
infrastructure (nest boxes, slats, perches...) has been installed. The effect of the age of the
infrastructure may be explained by the fact that the older the infrastructure becomes, the more
difficult it gets to achieve sufficient standards of cleaning due to the wear of the materials,
both of the production system and of the building itself, especially when it is taken into
account that the level of environmental contamination increases significantly during a
production cycle (Wales et al., 2007). The importance of such latent environmental sources of
infections should not be underestimated since Davies and Wray (1996) described the survival
of Salmonella in empty poultry houses for a period of 12 months, which implies the risk of
„transfer‟ of an infection between successive production cycles. This leads us to the second
significant risk factor: previous Salmonella shedding on the farm.
Previous Salmonella shedding
The odds to detect Salmonella are higher on farms where previous Salmonella shedding
has occurred, i.e. where Salmonella was detected in pooled faeces samples or bootswabs
collected in the framework of the official monitoring program. This is in accordance with the
studies of Gradel et al. (2004) and Carrique-Mas et al. (2009), stating that the major part of
Salmonella-infections on laying hen farms are not newly introduced on the farm but are the
result of re-introduction of the pathogen from the farm‟s environment.
The importance of sanitary measures is illustrated by the fact that absence of dry
cleaning in between successive production rounds was found to be a significant risk factor for
the presence of Salmonella. Dry cleaning is the mechanical removal of organic material
Chapter 7 143
(manure, dust, feed spills…) before the wet cleaning (using water) is carried out. A
remarkable observation in our field study was that only 82.2 % of the 292 sampled farms
carried out dry cleaning of the laying hen houses, whereas for wet cleaning and disinfection
this was 95.5 % and 92.5 % respectively, with large differences between the countries. In
Germany and Italy for instance more than 1/3 of the farms did not carry out dry cleaning
between production rounds. This constitutes a risk since the value of dry cleaning in between
production rounds is 2-fold: on one hand it is very useful to remove organic matter that can
harbour Salmonella. On the other hand it contributes to a more efficient disinfection since the
presence of considerable amounts of organic protective matter such as manure, dust and
spilled feed has an adverse effect on the efficacy of disinfection (Davies and Breslin, 2003;
Gradel et al., 2004).
These findings underline once again the necessity of sensibilization and informing farmers
about the importance of continuing efforts to achieve and maintain high levels of biosecurity
on a laying hen farm.
4. RISK FACTORS FOR SALMONELLA SHEDDING: INFLUENCE OF THE HOUSING
SYSTEM
Already in 2002, the results of a retrospective epidemiological study of Mølbak and
Neimann suggested that eggs derived from hens housed in conventional battery cages yielded
a higher risk for infection of humans with Salmonella Enteritidis compared to eggs derived
from hens housed in non-cage housing systems. This finding was corroborated by the results
of the EFSA baseline study from 2004 - 2005, both at the Community level (EFSA, 2007) and
at the level of the individual member states (Methner et al., 2006; Namata et al., 2008;
Huneau-Salaün et al., 2009). Also the results of our cross-sectional study in 292 laying hen
flocks identified the housing in conventional battery cages as a risk factor.
However, the observed influence of the housing type does not necessarily mean that
there is a causal relationship between the housing system and the level of Salmonella
excretion. On the contrary, it is more likely that the housing system is strongly entangled with
several other production characteristics such as the farm and the flock size, the age of the
building, previous Salmonella infections on the farm etc. Below, a number of laying hen
husbandry characteristics that may be related to both the housing system and the probability
of detecting Salmonella are discussed.
144 Chapter 7
First of all, the number of flocks on the farm and number of hens in a flock are shown
to be risk factors for Salmonella infections in laying hens, independently of housing type
(Heuvelink et al., 1999; Mollenhorst et al., 2005; EFSA, 2007; Snow et al., 2007; Carrique-
Mas et al., 2008; Huneau-Salaün et al., 2009). Several studies, including our cross-sectional
study, have shown that conventional battery cage farms are in general larger farms, not only
with more hens per flock but also with more flocks on the farm (EFSA, 2007; Carrique-Mas
et al., 2008; Van Hoorebeke et al., 2010). This could be one of the confounding factors
explaining why conventional battery cage farms are more frequently positive for Salmonella
than non-cage housing systems. The presence of multiple flocks on one farm may enhance
cross-contamination from one flock to another, especially when the different flocks and laying
hen houses on the farm are linked through egg conveyor belts, feed pipes, passageways etc.
(Carrique-Mas et al., 2008). Furthermore, as is often the case on farms with multiple flocks,
not all the hens are of the same age. Multistage production has also been identified as a risk
factor for Salmonella in laying hens (Mollenhorst et al., 2005; Wales et al., 2007; Carrique-
Mas et al., 2008; Huneau-Salaün et al., 2009).
Another factor which could play a role is the higher stocking density in conventional
battery cage housing compared to non-cage housing systems. For many infectious diseases in
production animals it has been demonstrated that a higher stocking density increases the
prevalence of disease and the ease of spread (Dewulf et al., 2007). With reference to
Salmonella, it has been shown in pigs that higher stocking densities increase the risk of
Salmonella infections (Funk et al., 2001; Nollet et al., 2004). However, to our knowledge no
results on this parameter are available regarding Salmonella infections in laying hens. Maybe
this is due to the fact that this parameter was never evaluated or that it has been studied but
never had been found to be significantly influential. Possibly the high density of laying hens
in conventional battery cages, and as a consequence the large volume of faeces and dust
increases the incidence of Salmonella infections in this type of housing (Davies and Breslin,
2004). High stocking densities could also indirectly interact with Salmonella infections
because of the stress caused. Yet, literature is not unequivocal on the influence of stocking
density on stress in laying hens and the relationship between stress and different housing
types (Craig et al., 1986; Koelkebeck et al., 1987; Davis et al., 2000).
The difference in ease to clean and disinfect the different housing systems is also an
important factor. In contrast to broiler and finishing pigs houses, which are in general
relatively simple structured, laying hen houses are notoriously difficult to clean and disinfect
Chapter 7 145
because of their intrinsically complicated structures (Wales et al., 2006). In particular
conventional battery cage systems are considered to be extremely hard to clean and disinfect
sufficiently because of the restricted access to cage interiors, feeders, egg belts, and so forth
(Davies and Breslin, 2003; Carrique-Mas et al., 2009a). It is thought that the big advantage of
non-cage systems is that in between production rounds the whole infrastructure (nest boxes,
slats, perches, feeding troughs...) can be dismantled and thoroughly cleaned and disinfected,
in contrast to batteries of cages which are never dismantled. However, based on the results of
our cross-sectional study, 21.2 % of the farmers keeping laying hens in non-cage systems do
not dismantle the infrastructure. This means that cleaning and disinfection of the sampled
house is carried out when the nest boxes etc. are still installed. It may be questionned whether
in such conditions a sufficient cleaning and disinfection of both the house and the
infrastructure is possible.
It has been suggested that conventional battery cage housing presents a more attractive
environment to pests because the laying hens can interfere less with their movements since
the birds are restrained to cages (Carrique-Mas et al., 2009b). Because of their role as vectors
in the transfer of Salmonella, various pests such as rodents, flies, wild birds etc. have been
extensively discussed (Guard-Petter, 2001; Davies and Breslin, 2003; Kinde et al., 2005;
Carrique-Mas et al., 2009a). In our study, the focus was on another widespread pest in laying
hens‟ houses: the poultry red mite (Dermanyssus gallinae).
Besides the direct physiological harmful effects on the hens, poultry red mites also
present a serious threat to health of the animals (Kilpinen, 2005): since long its role as a
vector for several diseases such as chicken pox virus, Newcastle virus, Salmonella
Pullorum/Gallinarum has been recognized (Zeman et al., 1982). The poultry red mite‟s role in
Salmonella infections in laying hens is three-fold. Firstly, mass red mite infestations can lead
to immunosuppression (Kowalski and Kosol, 2009), increasing the hen‟s susceptibility for
infections. Secondly, the presence of large numbers of red mites causes agitation and stress.
Finally, it has been reported under experimental conditions that mites could play a role in the
persistence of Salmonella in laying hens, either by transferring the bacterium from hen to hen
or by hens consuming contaminated mites leading to a persisting infection (Valiente-Moro et
al., 2007; Valiente-Moro et al., 2009). Information on the role of Dermanyssus gallinae as an
active vector for Salmonella under field conditions was scarce. Therefore it was decided to
include the bacteriological analysis of red mite samples in the cross-sectional field study.
146 Chapter 7
In total, in 214 of the 292 sampled houses red mites were collected. Concerning the
prevalence of red mites, no significant differences between the different housing types and the
different countries were observed. In fact, in none of the red mite samples Salmonella was
detected, not even in the flocks found positive for Salmonella. Whether this means that
Dermanyssus gallinae plays no role at all in the spread of Salmonella in a laying hen flock is
not yet clear. Further research is needed to elucidate this matter.
Finally, there is the influence of stress. Stress has been shown to have an
immunosuppressive effect in laying hens (El-Lethey et al., 2003; Humphrey, 2006), which
can have negative consequences with respect to Salmonella shedding. There are several
moments in the laying hen‟s life where the bird is subjected to stress: moving from the rearing
site to the egg producing plant (Hughes et al., 1989), the onset of lay (Jones and Ambali, 1987;
Humphrey, 2006), in the final stages of the production period, during thermal extremes
(Thaxton et al., 1974; Marshally et al., 2004) or transportation to the slaughterhouse (Beuving
and Vonder, 1978). Typical for laying hen husbandry is the practice of induced moulting. The
effects on S. Enteritidis infections during moult have been extensively studied: moulted hens
shed more S. Enteritidis in their eggs and faeces (Holt, 2003; Golden et al., 2008), have higher
levels of internal organs colonization (Holt et al., 1995) and exhibit more pathology in the
intestinal tract than non-moulted hens (Holt and Porter, 1992; Holt and Porter, 1993; Macri et
al., 1997). Furthermore, moulting causes the recurrence of previous S. Enteritidis infections
(Holt and Porter, 1993). There are some contradictory data on the influence of the housing
type on the stress levels in laying hens. Some studies suggest that laying hens have less stress
in conventional battery cages (Koelkebeck et al., 1986; Craig et al., 1996) whereas other
authors state that hens housed in non-cage systems experience less stress (Hansen et al., 1993;
Colson et al., 2008). With regard to the housing system, the age of the hens (Singh et al., 2009)
and the breed of the hens could also play a role: certain hen breeds exhibit significantly higher
stress responses when raised in deep litter versus free-range systems, compared to other
breeds (Campo et al., 2008). Based on the few studies exploring the stress response of hens
housed in different housing systems, no consistent conclusion on the influence of the housing
system can be drawn.
All this suggests that the nature of this topic is very complex and that a multi-level
approach is required to tackle the problem. In a first step it is necessary to control the
shedding of Salmonella. The housing system has an influence on this: the housing of laying
hens in conventional battery cages increases the risk of shedding Salmonella. In a further
Chapter 7 147
stage, the Salmonella infection on the farm, i.e. the actual presence of the pathogen in the
hens or the environment, will have to be controlled. This is much more difficult to achieve
because other factors than the commonly known ones (e.g. housing in battery cages, larger
flocks sizes, age of the hens…) seem to play a role. Strict and maintained biosecurity
measures and an optimized management will be the key to success.
5. ANTIMICROBIAL RESISTANCE IN INDICATOR BACTERIA IN LAYING HENS
It is often believed that the role of laying hens in the transfer of antimicrobial
resistance to humans is low compared to other animal species such as broilers, pigs and cattle.
This is based on the fact that antimicrobial usage is generally low in laying hens and that eggs
are thought not to be very suitable vectors for transfer of resistant bacteria to the consumer
(Yoshimura et al., 2000; Musgrove et al., 2006). However, the consumption of eggs and meat
of culled hens could pose a threat to public health if antimicrobial resistant bacteria would be
present. Moreover, differentiation between laying hens and broilers in monitoring programs
for poultry allows drawing conclusions on the selective pressure of antimicrobial usage.
Recent reports indicate that both in poultry and other animal species the move from
conventional indoor production towards free-range and organic production exerted a
beneficial effect on the levels of antimicrobial resistance in zoonotic and indicator bacteria
(Avrain et al., 2003; Thakur and Gebreyes, 2005; Ray et al., 2006, Schwaiger et al., 2008;
2010, Young et al., 2009). On the other hand it has been shown that this move to non-cage
housing systems for laying hens resulted in an increased incidence of particularly bacterial
diseases (Fossum et al., 2009; Kaufmann-Bart and Hoop, 2009), which could potentially lead
to increased antibiotic usage. Epidemiological data on antimicrobial resistance in laying hens
in different housing systems are scarce. Therefore, the levels of antimicrobial resistance in 2
indicator bacteria in laying hens were determined in chapter 6. Special attention was paid to
the influence of the housing system on antimicrobial resistance.
One of the most remarkable findings is the high prevalence of ceftiofur-resistant
isolates in Belgium (12 %) compared to the results of Switzerland, Italy and Germany where
0 %, 3 % and 4 % of the isolates were resistant. Although the level of ceftiofur-resistance in
Belgian laying hens is much lower than the levels observed in Belgian broilers (44 %)
(Persoons et al., 2010), it is necessary to sort out what are the causes of the emergence and
spread of the resistance against such a critically important antimicrobial.
148 Chapter 7
Although the problem of increasing Extended Spectrum Beta Lactamases-resistance is
not limited to Belgium, it is striking that in our study Belgian isolates showed significant
higher levels of ceftiofur-resistance. One of the reasons for the increasing levels of ceftiofur
resistance may be the worldwide and systematic use of ceftiofur in breeding eggs and one-
day-old chicks in the hatcheries (Collignon and Aarestrup, 2007; CMAJ, 2009; Webster,
2009). In Belgium the use of ceftiofur in poultry has not been licensed for a decade (Bertrand
et al., 2006) but might be continued off-label (Persoons et al., 2010). Another hypothesis for
the increased ceftiofur resistance might be the production on the same farm of other animal
species such as pigs and cattle, in which the use of ceftiofur is still registered. This could
result in horizontal transmission of resistance genes between different bacterial species (Kruse
and Sorum, 1994; Lorenz and Wackernaegel, 1994).
Concerning the influence of the housing system, the non-cage production systems for
laying hens did not show consistent results with regard to MDR in E. coli and E. faecalis. The
levels of MDR in E. faecalis were lower in free-range laying hens than in the conventional
battery cage system, whereas increased levels of MDR were seen in E. coli in floor-raised
hens.
Although the microbiological integrity of eggs still seems to be the most urgent food
safety aspect of laying hen husbandry, this study indicates that the issue of antimicrobial
resistance in laying hens should not be neglected. Our results provide an interesting snapshot
of the situation in laying hens. However, the limited amount of data makes it very hard to
assess the size of the problem, to evaluate the evolution in time and to take corrective steps.
Therefore, it would be very useful to include laying hens as a separate production entity in
resistance monitoring programs. Since laying hens can be considered as a reference
population with rare antimicrobial usage, continuous resistance monitoring in laying hens
could serve as a useful tool for the detection of trends in the distribution of antimicrobial
resistance. To maximize the impact and value of such programs, standardization of protocols
and reporting in all member states is necessary (Wallmann, 2006; WHO, 2008; EFSA, 2008).
This would guarantee a more focused and generalized approach of the problem.
One of the cornerstones of such control programs should be the strict registration of
the amounts of antibiotics used in agriculture. Indeed, the curative, prophylactic and
metaphylactic use of large amounts of antimicrobials is common practice in modern
husbandry of several food animal species (Aarestrup, 2004; EFSA, 2008) and concerns have
Chapter 7 149
grown that this usage raised antimicrobial resistance in animals and humans (Wegener et al.,
1999; Aarestrup, 2005; Acar and Moulin, 2006; Aminov and Mackie, 2007). Registration of
the amounts and types of antibiotics used and the epidemiological analysis of the relationship
between antimicrobial usage and the emergence and spread of resistance would allow to
further evaluate this link between antimicrobial usage and the prevalence of antimicrobial
resistance.
Even though it certainly has played a huge role in the development and spread of
antimicrobial resistance in animals and humans, the debate should not be narrowed down to a
yes-or-no argument on the –abundant− use of antibiotics in food animal production. There is
little doubt that the whole issue of antimicrobial resistance is more complex than that and that
factors such as the use of antibiotics in humans (Cizman et al., 2001), the spread of resistance
from companion animals to humans (Canton and Coque, 2006; Skurnik et al., 2006) and the
presence of resistant flora in diverse eco-systems such as surface water (Acar and Moulin,
2006; Moore et al., 2006; Servais and Passerat, 2009) also contribute to the complexity of the
problem. Further epidemiological and microbiological research is needed to better understand
the spread and mechanisms of antimicrobial resistance.
6. SPECIFIC TARGETS FOR FUTURE RESEARCH
This thesis has provided some useful information on the presence of Salmonella and
antimicrobial resistance on laying hen farms and the associated risk factors, with special
emphasis on the influence of the housing system. However, changing legislation and
increasingly critical consumers‟ demands will require continuous efforts and adaptations of
the laying hen sector. Scientific research can be very useful in this process, identifying factors
to fine tune and optimize the management of the farms and to provide guidelines for policy
makers.
Untill now, the focus of control programs in laying hens has been on S. Enteritidis and
S. Typhimurium. When in the foreseeable future a zero-prevalence of all Salmonella-serovars
may be required, additional or different measures will have to be taken. In order to increase
the efficiency of such measures, more epidemiological data on the prevalence of these
serovars and the risk factors associated with their presence must be available.
The strict monitoring of the prevalence of Salmonella in different housing systems
must continue. Further studies must be carried out to determine which sampling protocol is
150 Chapter 7
sensitive enough to provide accurate estimates of the prevalence in vaccinated flocks with low
levels of infection.
Although the antimicrobial resistance issue seems less outspoken in laying hens
compared to broilers, pigs and cattle, it would be useful to include laying hens in
antimicrobial resistance monitoring programs in order to assess the size of the problem and to
monitor the evolution in time. Special attention should be paid to the prevalence and spread of
β-lactam resistance in laying hens.
7. CONCLUDING REMARKS
Based on the results of the studies carried out during the current thesis, it is most
unlikely that the move from conventional battery cages towards non-cage housing systems
will increase the prevalence of Salmonella in laying hen flocks. However, it would be very
naïve to state that the ban on battery cages will be the answer to the well-known egg-related
Salmonella problem in public health. The apparent advantage of non-cage systems is most
probably influenced and biased by other factors such as the age of the infrastructure, flock
size and sanitary conditions.
Our results from the extensively sampled flocks clearly indicate that in a considerable
proportion of these vaccinated flocks Salmonella is still present in the hens, regardless of the
housing type. These findings suggest that if the decision would be taken to stop the mandatory
vaccination of commercial laying hens, the prevalence of Salmonella could rapidly increase
again, also in the non-cage housing systems.
Furthermore, there is the issue of the Salmonella serovars different from Enteritidis
and Typhimurium. The presence of such serovars does not seem to be influenced by the
housing type and vaccination strategy, suggesting that the control of these serovars will be
completely dependent on biosecurity and management strategies. This raises the question
whether the commonly accepted measures to control Salmonella will be adequate enough to
counter all serovars of Salmonella.
Concerning the antimicrobial resistance in laying hens, no consistent conclusion on the
influence of the housing system could be drawn for Escherichia coli and Enterococcus
faecalis. Compared to battery cages, the levels of multiple drug resistance were higher for E.
coli in floor-raised systems and lower for E. faecalis in free-range systems. Although these
Chapter 7 151
differences are statistically significant, it is not yet clear whether they have some biological
relevance.
152 Chapter 7
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SUMMARY
Summary 163
The housing of laying hens in conventional battery cages will be forbidden in the EU
from 1 January 2012 onwards. Council Directive 1999/74/EC states that from then onwards
the housing must restricted to enriched cages and non-cage housing systems. This ban on
conventional battery cages aims to improve the welfare of laying hens. Although the
influences of these alternatives for conventional battery cages on laying hen welfare,
productivity and user-friendliness have been extensively evaluated and discussed, it has also
initiated the question whether there are any adverse consequences of this decision on the
spread and/or persistence of infectious diseases in a flock. For instance the intensive and
frequent hen-to-hen contacts and the presence of litter in non-cage systems jeopardize the
biosecurity and increase the risk for specific diseases to develop and spread. This has led to
the question whether the same effect is to be expected for zoonotic pathogens.
One of these zoonotic agents is Salmonella, which is worldwide still an important
cause of human disease. In Europe, Salmonella Enteritidis and Salmonella Typhimurium are
the most commonly isolated serovars in human cases of salmonellosis and contaminated eggs
still remain the most important source of infection with Salmonella Enteritidis for humans.
Apart from the microbiological integrity of the eggs, the emergence and spread to
humans of antimicrobial resistance through the consumption of food from animal origin plays
a very important role (CHAPTER 1). The aims of this thesis were to determine the influence of
laying hen housing systems on the prevalence of Salmonella and antimicrobial resistance in
laying hens (CHAPTER 2).
In CHAPTER 3, the results of a study to determine whether the current sampling
protocols (i.e. a limited number of pooled faeces and / or dust samples) are sufficiently
sensitive to detect expected low within-flock prevalences of Salmonella are presented. In total,
19 flocks were sampled. All the sampled flocks were found negative for Salmonella based on
the results from the official Salmonella control program. Using an extended on-farm sampling
methodology (5 pooled faeces samples, 40 cloacal swabs of 40 hens and 1 mixed dust sample),
Salmonella could not be detected in any of the flocks. After transportation of the hens to the
laboratory and subsequent analysis of cloacal swabs and caecal contents, Salmonella
Enteritidis was detected in laying hens from 5 out of 19 farms. The observed within-flock
prevalence, based on the cloacal swabs, ranged from 1 – 14 %. The differences between the
samples taken on-farm and after transport may be explained by the intermittent excretion of
Salmonella by infected animals and the fact that stress, caused by the transport, may make
hens go from a „carrier‟ state to a „shedding‟ state. These results indicate that depending on
164 Summary
the sampling procedure different estimates of the between- and within-flock prevalence of
Salmonella can be obtained. Although postmortem examination of hens is labour-intensive
and expensive, the results of this study suggest that it is the best method for the detection of
Salmonella in low prevalence flocks.
Risk factors for Salmonella infections in laying hen flocks were identified in
CHAPTER 4. For this purpose 29 laying hen flocks, including farms using conventional and
alternative housing systems, were intensively sampled both on-farm and after transport as
described in chapter 3. An on-farm questionnaire was used to collect information on general
farm and flock characteristics (e.g. flock size, breed, age of the hens, medical treatments…)
and biosecurity measures. Salmonella was detected in laying hens from 6 of the 29 sampled
farms. A previous Salmonella contamination on the farm and the age of the production system
(the number of years that the current infrastructure is in use) were identified as risk factors for
Salmonella infections in laying hens. The results of this study indicate that, irrespective of the
housing system in which the hens are housed, persistent biosecurity measures are necessary in
order to prevent the recurrent contamination or new infections of laying hen flocks in
subsequent production cycles.
In the next study (CHAPTER 5), the aims were to determine the between- and within-
flock prevalence of Salmonella spp. in laying hen flocks housed in conventional battery cage
and alternative non-cage housing systems and to identify risk factors for the shedding of
Salmonella on laying hen farms. Therefore, a cross-sectional study was carried out in 5
European countries (Belgium, Germany, Greece, Italy and Switzerland). In total 292 laying
hen flocks were sampled in the month prior to depopulation. The on-farm questionnaire
described in chapter 4 was used to collect information on general management practices and
specific characteristics of the sampled flock. Twenty-nine flocks were found positive for at
least 1 Salmonella-serotype. In these flocks the within flock prevalence of shedding hens,
determined by individual sampling of 40 hens, varied between 0 % and 27.50 %. A wide
variety of serotypes was isolated with Salmonella Enteritidis as the most common. Housing in
conventional battery cages, the absence of dry cleaning in between production rounds and
sampling in winter turned out to be risk factors for the shedding of Salmonella Enteritidis or
Typhimurium. The results of this study illustrate that, despite the fact that in non-cage
housing systems the chance of oro-faecal transmission of Salmonella is much higher than in
conventional battery cage systems, no higher prevalence of Salmonella could be observed in
flocks housed in these alternative systems. Several management and biosecurity measures
Summary 165
such as strict cleaning and disinfection practices have been identified as protective factors to
minimize the introduction and persistence of Salmonella on laying hen farms.
In a last study (CHAPTER 6), the influence of the housing type on antimicrobial
resistance in Escherichia coli and Enterococcus faecalis from laying hens was determined. In
total, 1102 E. coli and 792 E. faecalis isolates were tested from 4 European countries
(Belgium, Germany, Italy and Switzerland). The isolates were described by multiple drug
resistance (MDR) patterns. Country and housing type displayed different influences on the
MDR levels of both species. In E. coli, the housing in a floor-raised system resulted in an
increased risk of MDR in comparison to the conventional battery system. In E. faecalis the
MDR levels were lower in free-range systems than in conventional battery cages. In Belgium,
ceftiofur-resistant E. coli isolates were more abundantly present than in the other countries.
The results of this study suggest that the levels of antimicrobial resistance in indicator bacteria
such as E. coli and E. faecalis in laying hen flocks are relatively low compared to the levels
observed in broilers and pigs. The differences observed between both indicator bacteria with
respect to the influence of the housing system suggest that it is important to not focus on a
sole bacterial species when trying to assess risk factors for antimicrobial resistance. It is
crucial to conscientiously monitor the prevalence and evolution in time of antimicrobial
resistance in laying hens, both during the rearing period and the production cycle, to be able
to detect early changes in antimicrobial resistance and to minimize the spread of resistant
bacteria to humans.
Based on the results of the studies carried out during the current thesis, it is most
unlikely that the move from conventional battery cages towards non-cage housing systems
will increase the prevalence of Salmonella in laying hen flocks. However, the apparent
advantage of non-cage systems is most probably influenced and biased by other factors such
as the age of the infrastructure, flock size and sanitary conditions. The results from the
extensively sampled flocks clearly indicate that in a considerable proportion of these
vaccinated flocks Salmonella is still present in the hens, regardless of the housing type,
suggesting that stopping of vaccination against Salmonella of commercial laying hens could
rapidly lead to an increase of the prevalence. Concerning the antimicrobial resistance in
laying hens, no consistent conclusion on the influence of the housing system could be drawn
for Escherichia coli and Enterococcus faecalis (CHAPTER 7).
SAMENVATTING
Samenvatting 169
Het huisvesten van leghennen in batterijkooien zal in de gehele Europese Unie
verboden worden vanaf 1 januari 2012. De Europese Richtlijn 1999/74/EC houdt in dat vanaf
deze datum leghennen enkel nog in verrijkte kooien en niet-kooisystemen mogen gehuisvest
worden. Dit verbod heeft tot doel het welzijn van de leghennen te verbeteren.
Niettegenstaande het feit dat de invloed van deze alternatieven voor batterijkooien op het
dierenwelzijn, de productiviteit en de gebruiksvriendelijkheid uitvoerig onderzocht en
beschreven zijn, roept het tezelfdertijd de vraag op of er geen nadelige gevolgen zijn op het
vlak van de verspreiding en/of het aanwezig blijven van bepaalde infectieziektes in een toom.
De frequente en intense contacten tussen de dieren onderling en de aanwezigheid van strooisel
in niet-kooisystemen hypothekeren de bioveiligheid en verhogen het risico op het ontstaan en
de snelle verspreiding van bepaalde ziektes. De vraag kan gesteld worden of het zelfde kan
verwacht worden voor zoönotische agentia.
Eén van deze pathogenen is Salmonella, wereldwijd nog steeds een belangrijke
ziekteverwekker bij de mens. In Europa zijn Salmonella Enteritidis en Typhimurium de
vaakst geïsoleerde serotypes bij gevallen van humane salmonellosis. Voor de mens blijven
besmette eieren de belangrijkste bron van infectie met Salmonella Enteritidis.
Naast de microbiologische integriteit van de eieren is ook de verspreiding van
antibioticumresistentie naar de mens door de consumptie van voedsel van dierlijke oorsprong
een heel belangrijk facet van de voedselveiligheid (HOOFDSTUK 1). De doelstellingen van
deze thesis waren de effecten van huisvestingssystemen op de prevalentie van Salmonella en
antibioticumresistentie in leghennen na te gaan (HOOFDSTUK 2).
In HOOFDSTUK 3 worden de resultaten beschreven van een studie waarin werd
nagegaan of de huidige bemonsteringsprotocols (i.e. een beperkt aantal mengmeststalen al dan
niet gecombineerd met een stofstaal) accuraat genoeg zijn om op toomniveau lage
Salmonella-prevalenties te kunnen detecteren. In totaal werden 19 tomen bemonsterd. Al deze
tomen waren op basis van het officiële controleprogramma Salmonella-negatief bevonden.
Ook na een intensieve bemonstering (i.e. 5 mengmeststalen, 40 cloacaswabs van 40 hennen
en 1 stofstaal) op het bedrijf kon in geen enkel van de bemonsterde tomen Salmonella
teruggevonden worden. Na transport van 100 hennen naar het labo en daaropvolgende analyse
van cloacaswabs en caecale inhoud werd Salmonella Enteritidis teruggevonden in leghennen
van 5 tomen. Op basis van de cloacaswabs varieerde de prevalentie binnen een toom van 1 tot
14 %. Het verschillende resultaat van de staalnames op het bedrijf zelf en na transport kan
verklaard worden door de intermitterende excretie van Salmonella door geïnfecteerde dieren
170 Samenvatting
en het feit dat de stress die veroorzaakt wordt door het transport de dieren van een „drager
status‟ naar een „excreterende status‟ doet overgaan. Deze resultaten tonen aan dat afhankelijk
van de gebruikte bemonsteringsschema‟s verschillende schattingen van de Salmonella-
prevalentie worden bekomen. De resultaten van deze studie wijzen er op dat post mortem
onderzoek van leghennen de beste methode is om Salmonella te detecteren, hoewel het een
arbeidsintensieve en bijgevolg dure methode is.
Risicofactoren voor Salmonella-infecties werden geïdentificeerd in HOOFDSTUK 4.
Negenentwintig tomen, gehuisvest in batterijkooien en niet-kooisystemen werden intensief
bemonsterd zoals beschreven in Hoofdstuk 3. Tijdens het bedrijfsbezoek werd een vragenlijst
ingevuld om informatie te bekomen over bedrijfs- en toomeigenschappen (bv. de toomgrootte,
het ras, de leeftijd van de hennen, behandelingen van de toom,…) en de
bioveiligheidsmaatregelen. Salmonella werd teruggevonden in hennen uit 6 van de 29
bemonsterde tomen. Uit de risicofactoren-analyse bleek dat een voorgaande Salmonella-
besmetting op het bedrijf en de leeftijd van het productiesysteem (i.e. het aantal jaren dat de
huidige installaties in dienst zijn) een significante invloed hebben op de kans op Salmonella-
besmetting in leghennen. De resultaten van deze studie tonen aan dat, ongeacht het
huisvestingssysteem, strikte en aanhoudende bioveiligheidsmaatregelen nodig zijn om nieuwe
infecties of heropflakkeren van bestaande infecties te voorkomen tijdens opeenvolgende
productierondes.
In een volgende studie (HOOFDSTUK 5) werd de prevalentie van Salmonella-
uitscheiding nagegaan op leghennenbedrijven met verschillende huisvestingssystemen en
werden risicofactoren voor de uitscheiding van Salmonella geïdentificeerd. Er werd in 5
Europese landen een cross-sectionele veldstudie uitgevoerd: België, Duitsland, Griekenland,
Italië en Zwitserland. In totaal werden 292 leghennentomen bemonsterd de maand
voorafgaand aan depopulatie. De vragenlijst die beschreven werd in Hoofdstuk 4 werd
gebruikt om informatie te verzamelen over het algemene management op het bedrijf en
specifieke eigenschappen van de bemonsterde toom. In 29 van de bemonsterde tomen werd
tenminste 1 Salmonella-serotype gevonden. Gebaseerd op de cloacaswabs van 40 hennen
varieerde de prevalentie van Salmonella-uitscheidende hennen tussen 0 en 27.5 %. Er werden
verschillende Salmonella-serotypes geïsoleerd hoewel S. Enteritidis toch de meest
voorkomende was. De volgende risicofactoren voor de uitscheiding van Salmonella
Enteritidis/Typhimurium werden geïdentificeerd: het houden van leghennen in batterijkooien,
het ontbreken van droge reiniging tussen twee productierondes in en het bemonsteren tijdens
Samenvatting 171
de winterperiode. Er werd dus geen hogere Salmonella-prevalentie gevonden in tomen die
niet in batterijkooien gehouden werden, niettegenstaande in niet-kooisystemen de kans op de
oro-faecale overdracht van Salmonella tussen de dieren veel groter is. Verscheidene
managements- en bioveiligheidsmaateregelen zoals een strikte reiniging en disinfectie werden
geïdentificeerd als factoren die de insleep en verspreiding van Salmonella op een
leghennenbedrijf kunnen helpen minimaliseren.
In een laatste studie (HOOFDSTUK 6) werd de invloed van het huisvestingssysteem op
het voorkomen van antibioticumresistentie in Escherichia coli en Enterococcus faecalis
onderzocht. In totaal werden 1102 E. coli en 792 E. faecalis isolaten van leghennen verzameld
in 4 Europese landen (België, Duitsland, Italië en Zwitserland). De isolaten werden
beschreven aan de hand van multiple drug resistance (MDR) patronen. Uit de risicofactoren
analyse bleek dat land en huisvestingstype een invloed uitoefenen op de MDR in beide
bacterie species. In vergelijking met batterijkooien vergrootte het huisvesten van hennen in
scharrelsystemen het risico op hoge MDR niveaus in E. coli. Voor E. faecalis waren de
geobserveerde MDR niveaus lager in hennen gehuisvest in vrije uitloop-systemen vergeleken
met hennen in batterijkooien. In België werden significant meer ceftiofur-resistente E. coli
isolaten gevonden dan in de andere landen. De resultaten van deze studie tonen aan dat de
niveaus van antibioticumresistentie in indicatorbacteriën in leghennen relatief laag zijn in
vergelijking met de situatie bij andere diersoorten zoals braadkippen en varkens. De
verschillende invloed van de huisvesting op de graad van antibioticumresistentie in beide
indicatorbacteriën duidt erop dat het belangrijk is om niet op één enkele bacteriesoort te
focussen wanneer men risicofactoren voor antibioticumresistentie wil identificeren. Het is
noodzakelijk om zowel tijdens de opfokperiode als tijdens de productiecyclus de prevalentie
en de evolutie in de tijd van antibioticumresistentie in leghennen zorgvuldig te monitoren om
in staat te zijn veranderingen snel te detecteren en om indien nodig in te grijpen teneinde de
verspreiding van resistentie naar de mens te minimaliseren.
Gebaseerd op de resultaten beschreven in deze thesis kan geconcludeerd worden dat
het hoogst onwaarschijnlijk is dat het huisvesten van leghennen in niet-kooisystemen de
prevalentie van Salmonella zal doen toenemen. Desondanks moet men er zich toch bewust
van zijn dat dit schijnbare voordeel van de niet-kooisystemen meer dan waarschijnlijk
beïnvloed en vertekend wordt door andere factoren zoals de leeftijd van de infrastructuur, de
toomgrootte en hygiënestatus. De resultaten van de intensief bemonsterde tomen tonen
duidelijk aan dat in een aanzienlijk deel van de gevaccineerde tomen Salmonella nog steeds
172 Samenvatting
aanwezig is, ongeacht het huisvestingstype. Dit wijst er op dat het afschaffen van de
vaccinatie van leghennen snel zou kunnen leiden tot een stijging van de prevalentie. Wat de
antibioticumresistentie betreft kon geen éénduidige conclusie getrokken worden voor
Escherichia coli en Enterococcus faecalis wat betreft de invloed van het huisvestingsysteem.
(HOOFDSTUK 7).
CURRICULUM VITAE
Curriculum Vitae 177
Sebastiaan Van Hoorebeke werd geboren op 4 februari 1980 te Gent. Na het behalen
van het diploma hoger secundair onderwijs aan het Don Boscocollege te Zwijnaarde (richting
Grieks-Latijn) werd de studie Diergeneeskunde te Gent aangevat. Hij behaalde het diploma
dierenarts (optie Varken-Konijn-Pluimvee) in 2006 met onderscheiding.
In oktober 2006 trad hij in dienst van de Vakgroep Voortplanting, Verloskunde en
Bedrijfsdiergeneeskunde als doctoraatsbursaal met een driejarig mandaat gefinancierd door de
Europese Unie. Hij verrichtte een onderzoeksstudie over de prevalentie van Salmonella en
antibioticumresistentie bij leghennen in verschillende huisvestingssystemen. Vanaf 1 oktober
2009 was hij aan de zelfde vakgroep werkzaam op een éénjarig FOD-project over Salmonella
Gallinarum.
Gedurende de 4 jaren aan de vakgroep was hij ook actief in de bedrijfsbegeleiding
Varken en stond hij mee in voor de opleiding van de optievakkers. Hij nam eveneens deel aan
de nacht- en weekenddiensten van de kliniek verloskunde Rund.
Sebastiaan Van Hoorebeke is auteur of mede-auteur van meerdere wetenschappelijke
publicaties in internationale tijdschriften en was spreker op verschillende nationale en
internationale congressen.
DANKWOORD
DANKWOORD
Weggemoffeld in de cloaca van dit boekje vinden jullie dit dankwoord.
Elke gelijkenis met bestaande personen berust louter op de werkelijkheid.
“Noblesse oblige” en daarom begin ik graag met mijn beide promotoren: Prof. Dr. Dewulf en Prof. Dr.
Van Immerseel. Jeroen, hartelijk dank voor je blijvende geloof in dit onderzoek, ook als ik een andere
richting uitstuiterde. Het was af en toe eens stevig zweten als een nieuwe deadline me bijna bij de
lurven had maar vandaag ben ik er helemaal van overtuigd dat het dubbel en dik de moeite waard is
geweest, bedankt. Filip, je heel deskundige en immer flegmatieke input waren van onmisbare waarde
in de voortgang van dit onderzoek. Het is echt een luxe om zulk een autoriteit als promotor te hebben,
of het nu over Salmonella of Clostridium gaat!
Ook een hartelijke dank aan Prof. Dr. Ducatelle. Vanaf het prille begin was u heel nauw betrokken bij
het Safehouse-project. Dank voor uw enthousiaste, niet aflatende interesse in mijn onderzoek.
Prof. Dr. Haesebrouck, bedankt dat ik gebruik mocht maken van uw labo‟s en de snelle en accurate
verbeteringen van mijn manuscripten.
Ten slotte ook nog mijn welgemeende dank aan Prof. Dr. Pasmans (Frank) voor de ijver waarmee je
mijn wetenschappelijke kattebelletjes las en corrigeerde.
Ik wil ook nogmaals van harte de leden van de begeleidings- en examencommissie bedanken voor de
tijd en moeite die ze staken in het nalezen en corrigeren van mijn thesis: Dr. Lieve Herman, Prof. Dr.
Berge, Dr. Hilde Van Meirhaeghe, Dr. Yves Van der Stede, Prof. Dr. De Zutter en Prof. Dr. Ducatelle.
Jantina, jij en ik waren de „jonge wolven‟ van het Belgische Safehouse-luik. Bedankt voor alle hulp bij
de vele euthanasies en labo-analyses. Veel succes en plezier in je nieuwe job!
Ook een woord van dank voor de mensen van Degudap voor het aanleveren van een heleboel
leghenbedrijven die wilden deelnemen aan ons onderzoek.
Het bemonsteren van al die bedrijven is één ding, bij aankomst in het labo begint het echte werk. BPW
bereiden, media maken, platen gieten, overenten… Daarom mijn zeer grote dank aan Sophie Callens,
Sofie Haerens en Lonneke Verbeek voor het vele werk in het labo.
To all the partners of the Safehouse-project: thank you very much for the nice cooperation. It was a
wonderful experience to work with people from so many different countries and institutions. The
yearly meetings always were very productive and enjoyable occurrences.
Johan Z (Proefbedrijf Pluimveehouderij): hartelijk dank voor uw interesse in mijn onderzoek en de
toelating om ook op uw bedrijf stalen te mogen nemen.
Marc V, je volgde van in het begin met veel belangstelling de voortgang van mijn onderzoek, het was
dan ook een aangename verrassing toen we plots „facultaire‟ collega‟s werden.
Na 3 jaar Safehouse kwam Salmopoul, hetgeen me opnieuw de kans gaf om enkele fijne mensen te
ontmoeten. Hilde VM, je rechttoe-rechtaan aanpak was heel verfrissend en motiverend. Ik hoop dat we
in de toekomst nog zullen kunnen samenwerken. Mieke G., veel succes met je bestrijding van Java en
consorten! Isabelle DW, Koen DR en Marc H. (ILVO): ik ben jullie heel dankbaar voor alle knowhow
en de uitvoering van de typeringen. Johan VE (Galluvet): hartelijke dank voor het aanleveren van de te
bemonsteren bedrijven en relevante praktijkkennis allerhande.
Koen P, heel hartelijk dank voor het kunstwerk dat de kaft van dit boekje siert!
Hoewel ik officieel tot de DI08 behoorde, speelde veel van de actie zich ook af op de vakgroep
Bacterio, Patho en Pluimveeziekten. En hoewel ik een koekoeksjong was heb ik absoluut nooit te
klagen gehad over de ontvangst, zélfs niet als ik het ganse gebouw murw sloeg met een bombardement
van vloeibare en op lichaamstemperatuur gebrachte kippenmest. Daarom een hele grote dankjewel aan
iedereen voor alle uitleg, de leuke babbels en de prettige „buitenschoolse activiteiten‟.
Echte mannen doen aan sport, dat weet iedereen. Daarom wil ik uitdrukkelijk de Verbancksjes danken,
de voetbalploeg waar een goed resultaat als een leuk extraatje wordt beschouwd. Jurgen, Gunter,
Patrick V, Jo DW, Ward, Alexander, Serge, Koen en Baptist: hartelijk dank voor de zeer fijne
sportieve en para-sportieve uitspattingen op en naast het veld. Ook dank aan de moedige collega‟s die
de sportieve strijd met de studenten aangingen: Bart P., Peter DS, Hans D, Miguel (always a pleasure!),
Stefanie S, Tamara L, Johnny V, Marc G. en Ruben VG.
Prof. Dr. de Kruif, dank voor uw interesse in mijn onderzoek, zowel wat de wetenschappelijke
voortgang als de meer praktijkgerichte benadering ervan betreft.
De vakgroep Voortplanting, Verloskunde en Bedrijfsdiergeneeskunde: een rovershol waarnaar het
altijd prettig terugkeren was na één of andere wetenschappelijk verantwoorde veroveringstocht. Het
echte hoofdkwartier is zonder de minste twijfel „den 48‟. Stefaan, een heel hartelijke dankjewel voor
alles wat je hebt gedaan voor mij, in de soms woelige omstandigheden. Of het nu over epidemiologie
ging, of over gecompliceerde keizersnedes of over allerhande andere chaotische en intense toestanden:
je hulp is van onschatbaar belang geweest. Ik wens je het allerbeste toe in alles wat je wilt en doet.
Davy, we hadden dat prima naar ons zin, zo op den 48. Merci voor alle leute, alle hulp (met de
computer en alle mogelijke praktische calamiteiten) en alle gesprekken. Veel succes ook nog met je
(post)doc! Ge weet het: “ge moogt nooit zeggen dat ge iets nie lust als ge‟t nog nie geproefd hebt”. En
dan Sarah. Ons gemeenschappelijke wetenschappelijke carrière heeft maar een tweetal jaar geduurd
maar het was een fijne tijd (“Avez-vous une sasse hygiénic?)”. Ik memde over het buitenland en werk
nu in Deinze, jij memde over het buitenland en werkt nu tussen de glooiende heuvels van Rwanda…
Ik vind het chique dat jullie die stap durfden zetten, heel veel succes gewenst! My dear Alfonso, I like
you so much that you‟re mentioned twice in this dankwoord. We shared a lot of memorable moments,
both in the context of Science and in „other contexts‟. You‟re a fantastic guy. De volgende in de rij is
Loes, die een stevige portie Hollandse bravoure toevoegde aan den 48. Je heerlijke no-nonsense stijl
was een hele verrijking en je bent het levende bewijs dat er vrouwen bestaan die kunnen meepraten
over epidemiologie én over voetbal (weliswaar over de verkeerde ploeg maar goed). Bénédicte, ik heb
nooit het geluk gehad om samen met jou den 48 te bevolken maar na het voorbije jaar (en Nantes) ben
ik er heel erg van overtuigd dat je er helemaal thuis hoort (“Parlez-vous français?”).
Naast het E-team had ik het genoegen om deel uit te maken van een andere sub-unit, zijnde
Bedrijfsdiergeneeskunde Varken. Prof. Dr. Maes, Dominiek, hartelijk bedankt voor de fijne
samenwerking en het vertrouwen gedurende de voorbije 4 jaren. Caroline, bedankt voor al je input
ivm bedrijfsproblemen en het bijhorende gebabbel. Querido Alfonso i Rubén, fue un gran placer de
conocer vos. The both of you are really een verrijking for the pig unit, I wish you all the best with your
research. Espero que nos volveremos a encontrar pronto, para tomar unas cervezas con vosotros.
Emily, Ellen, Josine, Liesbet, Iris V. en An C: bedankt voor de vlotte samenwerking én de
wetenschappelijke input gedurende de journal clubs!
Bart M. en Tom M., jullie zijn diegenen die me als student en als beginnend bursaaltje de kneepjes van
de bedrijfsdiergeneeskunde hebben bijgebracht, bedankt hiervoor.
Philip V., hartelijk dank voor de interesse in mijn onderzoek, de verhelderende gesprekken en de
nuttige feedback bij bedrijfsproblemen.
Tamara VDS, ik heb de varkens nu wel verlaten maar ik weet zeker dat we elkaar zeker nog zullen
ontmoeten.
En uiteraard moet ik ook de varkenshouders bedanken voor de aangename samenwerking. Een zeer
dikke merci aan Christian en Isabelle B., Herman en Dina L., Jim, Geert en Lydia (Agrivet) en Kristof
D., Hans, Bart, Jan en Jos (ILVO). De bedrijfsbezoeken op het ILVO brachten me ook in contact met
Sam M. en Marijke A. Sam, bedankt voor je interesse in mijn onderzoek en je kijk op het onderzoek.
Marijke, het allerbeste met jouw verder onderzoek en carrière.
Nadine, we hebben alle twee reeds de faculteit verlaten maar het is altijd fijn om je terug te zien.
Bedankt voor de zeer fijne tijd. Ria en Els, Marnik en Véro, Willy, Dirk en Wilfried: wat zou het zijn
zonder jullie? Een supermerci voor alle praktische hulp in de apotheek, de kliniek en de stallen, het
transport van de varkens, de ontelbare babbels en verfrissinkjes! Steven, dankjewel voor het geduld
dat je had met mijn „digibeet-zijn‟. En voor de gesprekjes natuurlijk. Sandra en Leïla, dankjewel voor
het regelen en controleren van alle mogelijke en onmogelijke (financiële) administratie. Nicole, je hebt
soms het genoegen (?) gehad om me ‟s morgens uit mijn slaap te halen. Heel erg bedankt voor al je
werk om de vakgroep en de rest van de faculteit netjes te houden, al zal ik wel altijd een sloddervos
blijven.
Geen inspanning zonder ontspanning. Gelukkig hadden we daar koffiepauzes, middagpauzes, terrasjes
en een ski-reis voor. Aan mijn prachtcollega‟s Muriel (“There‟s a crack in everything…”), Leen,
Josine, Iris, Isabel, Petra, Vanessa, Eline, Hilde en Lars: bedankt voor alle leuke gesprekken en de
gezelligheid! Voor diegenen die nog bezig zijn aan hun onderzoek: succes.
Ik heb een bloedhekel aan paarden maar dat neemt niet weg dat ik de collega‟s van Paard heel erg op
prijs stel. Te beginnen met Jan, Maarten en Catharina. Eerst en vooral dankjewel voor alle nachten dat
jullie tijdens die allereerste maanden ons, de beginnende assistenten, de keizersneden aanleerden.
Daarnaast -en vooral- ook een hele grote merci voor alle leute. Ik wens jullie nog heel veel succes met
het voltooien van jullie onderzoek! Katrien, daarmee zit het grote doctoraatsavontuur er bijna op. Wie
had dat ook alweer 4 jaar terug kunnen bedenken? Nog heel veel succes met alle toekomstige
avonturen! (nooit vergeten: surfen en paardrijden is voor snobs). Kim, jij ook nog veel succes met je
residency. Je was een supercollega.
Mocht Vlaanderen ooit ingelijfd worden bij Nederland dan is het beter om goed voorbereid te zijn.
Daarom heb ik altijd met veel plezier samengewerkt met mijn Hollandse collega‟s Iris, Cyrillus en
Wendy. Iris, het is dan toch geen Varken geworden voor mij (maar kalkoen is ook niet slecht!). Je bent
heel hard bedankt voor alle leuke momenten op de faculteit en daarbuiten. Veel succes met je verdere
carrière! Cyrillus en Wendy, onze samenwerking op de faculteit is van korte duur geweest maar
daarom niet minder aangenaam. Allebei veel succes gewenst in jullie nieuwe job. Ik neem aan dat we
elkaar regelmatig zullen blijven tegenkomen, hetgeen fijn is.
Jef en Marcel, de buitenpraktijk Rund heb ik nooit „gedaan‟ maar dat neemt niet weg dat ik steeds met
veel plezier naar jullie verhalen over het Echte Werk (vroeger en nu) kon luisteren. Ook de andere
collega‟s van Rund (Stefaan, Iris, Miel, Sofie, Karlien) wil ik bedanken om te komen depanneren als
een sectio op de kliniek een tikje te gecompliceerd bleek. Tot slot ook nog dank aan de overige
collega‟s van de vakgroep voor de prettige samenwerking.
Ik ben natuurlijk niet de eerste die de vakgroep verlaat. Enkele fantastische vrouwen zijn me
voorgegaan: Mirjan, Jo B (nog steeds met voorsprong mijn favoriete bio-ingenieuse), Nele G, Nele E,
Emilie VH en Leen M. Bedankt voor alle leuke momenten op en buiten de faculteit. Het is altijd fijn
om jullie terug te zien. Ook fantastisch doch veel minder vrouwelijk zijn de éminences grises die de
faculteit verlieten net toen ik er toekwam: Jo L, Boudewijn C, Geert H en Steven V. Jullie waren op
vele manieren een voorbeeld voor velen van de jonkies.
Niettegenstaande ik met heel veel plezier terugkijk op mijn periode aan de faculteit, ben ik toch heel
tevreden dat ik de stap heb gezet naar Quartes (Versele-Laga). Laat er geen twijfel over bestaan:
kalkoenen zijn de nobelste aller hoenders! Daarom gaat mijn dank in de eerste plaats uit naar Eric H,
Patrick Z en Ann V om me wegwijs te maken in „all things turkey‟. Daarnaast ook woorden van dank
voor P. Galle, Eveline DW en de overige collega‟s “in de villa”. Tot slot nog een hartelijke groet aan
K. De Praetere en Philippe DL van Volys Star.
Mijn ouders wil ik bedanken voor de prachtige jeugd die ik mocht beleven, een gegeven dat ik steeds
meer naar waarde ben gaan schatten. Ook toen alles anders werd kon ik steeds op jullie blijven
rekenen.
Beebs, hier sta ik dan. Superbedankt voor je eeuwige supporteren en je praktische regeltalent.
Uiteraard blijf je mijn volledige steun hebben bij de uitbouw van de vzw. Bomma, ik hoop dat de
eetavondjes nog lang mogen blijven duren. Ook jij bedankt voor het duimen.
Liesbeth (ofte Pr*t voor de vrienden), ook voor jou een grote dankjewel. Nu het doctoraat gedaan is
maak ik met plezier mijn belofte waar: dat wordt dus een dagje Boudewijnpark! En ik weet zeker dat
het „begeleid wonen-project‟ zal slagen. Ik ben er trots op dat ik je broer ben.
Paps en Alice, ook jullie heel hartelijk bedankt voor alle leuke en gezellige momenten en de
onvoorwaardelijke steun. Paps, met betrekking tot het trauma van Biochemie (2de
kan): bij deze beloof
ik je plechtig dat ik je nooit meer vraag om een cursus te onderlijnen exact 14 u vóór het examen.
Virginie, Thierry, Bernard, Yannick, Marjorie, Tom, Fanny, Geoff, Claire, Sébastien, Sarah en Fleur:
we liepen elkaar toevallig tegen het lijf in de “Nieuw samengestelde gezinnen”-statistieken en het mag
gezegd: ik ben heel blij dat ik jullie heb leren kennen. We hebben al bijzonder veel pret gemaakt en
dat zal in de toekomst niet anders zijn.
Het klassieke beeld van Dé Dierenarts wankelt en daar hebben de volgende zeer fijnbesnaarde
personen veel mee te maken: Hans en Philippe, jullie zijn schitterende vrienden. Ontelbare uren
hebben we reeds zitten zeuren, schateren of grienen, een cola of pilsje in onze knuistjes gekneld en dat
mag voor mijn part nog zeer lang blijven duren. Bedankt om er steeds te zijn als er „even top-overleg
moet gepleegd worden‟. Steven C, jij hebt niet kunnen weerstaan aan de lokroep van het Grote Geld
maar het is je vergeven. Je bent één van de meest onkreukbare mensen die ik ken en dat apprecieer ik
heel erg. Daarnaast denk ik met plezier aan onze bier- en frietensessies en de bijhorende gesprekken
terug. Diedrich, het is een waar genoegen om nog steeds contact te houden met jou. Waar is de tijd dat
we als schriele kuikens zaten te piepen dat “het nog allemaal moest beginnen”? Frederik VH, je bent
voor de eigen praktijk aan het gaan. Respect daarvoor! Ik heb enkele schitterende casussen, dus zeer
graag kortelings een wetenschappelijk symposium organiseren, wat denkt u?
Aldwin, Godfried, Hendrik, Jonas, Pieter C. en Pieter W: het mag heel duidelijk zijn waarvoor ik jullie
bedank: voor de jarenlange vriendschap, de heerlijke tijd in de Ottergemsesteenweg en de Friedrich
Froëbelstraat en de ontelbare momenten met een lach en een traan. De posse is uitgebreid met
vrouwen en kinderen maar ons vermogen tot volslagen debiliteit blijft godzijdank onaangetast. Jullie
zijn flinke knapen!
Toen het penthouse in de Friedrich Froëbelstraat was uitgebrand moest er een nieuw onderkomen
gevonden worden. Dan maar de Stropstraat. En ook daar heb ik mij vré goe gehad. Miel, ik bedacht
zonet dat we melkander zowaar bijna 10 jaar kennen, een periode waarin het aantal waanzinnige
toestanden en interessante feestjes moeilijk te tellen is. Het zijn alleszins prachtige herinneringen. Tuur,
plots stonden we met onze hele inboedel bij jou op kot. En we hebben er een fantastisch jaar van
gemaakt! Ik vind het chique hoe jij je weg zoekt binnen de machtige praktijkwereld, mijn
onvoorwaardelijke steun heb je alvast. En tot slot… ons Cumrolientje, het enige echte Orakel van
Leke. We zijn nu verschillende jaren verder en ook jij bent intussen aan de weg aan het timmeren, veel
succes! Wie weet komt er dra een reünie van de Stropstraat?
Maarten H, toen ik plots „dakloos‟ was bood jij de bovenverdieping in Balegem aan. Ik besef dat het
niet mijn meest constructieve periode was maar bij deze wil ik je uitdrukkelijk bedanken voor je
gastvrijheid en alle goeie zorgen. Ik hoop dat je in de job terechtkomt die je wilt, je verdient het
dubbel en dik.
Vincent, we plegen van tijd tot tijd nog eens te mijmeren over die tijd dat we als hevig puberende
rebellen het mooie weer maakten op ons aller Don Boscocollege. We‟ve come a long way... En het
stemt me bijzonder gelukkig dat we nog steeds het mooie weer kunnen blijven maken (“Als zelfs Fidel
Castro en de paus…”). Lynn, de tijd van de zebrabril ligt ook alweer ver achter jou. Ik ben erg blij dat
we elkaar hebben leren kennen. Ik wens jullie samen het allerbeste toe. Aan de kleine Jeff: tot dra!
Ik kus m‟n beide handjes want ik ken nog enkele schitterende mensen die ik heel erg apprecieer!
Daarom een dikke merci aan Pieter M, Debby, Frederief, Mandy (Phil Collins!), Fleur M, Wouter B.,
Filips, Lin, Wimpel, Valerie en Leen S.
David en Virginia, Jeanne en Jaak: hartelijk dank voor de gastvrijheid, de gezelligheid en de lekkere
etentjes.
En om te eindigen…
Venessa, mijn liefste Pluim, niemand is zijn of haar plaatsje in dit dankwoord zo hard waard als jij.
Bedankt voor alles, ik vind je fantastisch (en ik heb nog gelijk ook!). Bedankt voor wie je bent en wat
je betekent voor mij. Het heden met jou is al super, en de toekomst ziet er zelfs nog beter uit. Je weet:
ik ben een Handige Harry én een computerwizzard dus dat droomhuis en de layout van jouw boekje
komen voor elkaar!
Hoe graag ik je zie? Superveel graag!!
“And in the end
the love you take
is equal
to the love you make”
(The Beatles)
BIBLIOGRAPHY
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Van Hoorebeke S., Van Immerseel F., De Vylder J., Ducatelle R., Haesebrouck F., Pasmans
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infections in laying hens, with special emphasis on the housing system. XIXth European
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of Eggs and Egg Products, June 21st-25th 2009, Turku.
Van Hoorebeke S., Van Immerseel F., De Vylder J., Ducatelle R., Haesebrouck F., Pasmans
F., de Kruif A. and Dewulf J., 2009. Prevalence of antimicrobial resistance among
Escherichia coli and Enterococcus faecalis in Belgian laying hens. XIIth International Society
of Veterinary Epidemiology and Economics Conference, Durban.
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laying hens housed in conventional and alternative housing systems. XIIth International
Society of Veterinary Epidemiology and Economics Conference, Durban.
