Prevalence and Risk Factors of Enteropathogenic Yersinia ... · 2. Characteristics of Yersinia spp....
Transcript of Prevalence and Risk Factors of Enteropathogenic Yersinia ... · 2. Characteristics of Yersinia spp....
GENERAL INTRODUCTION
Prevalence and Risk Factors of Enteropathogenic Yersinia spp. in Pigs
at Slaughter Age
Gerty Vanantwerpen
Dissertation submitted in fulfillment of the requirements for
the degree of Doctor of Philosophy (PhD)
2014
Promoters:
Prof. Dr. Lieven De Zutter
Prof. Dr. Kurt Houf
Department of Veterinary Public Health and Food Safety
Faculty of Veterinary Medicine
Ghent University
Copyright
The author and the promoters give the authorization to consult and to copy parts of this
work for personal use only. Any other use is limited by the Laws of Copyright. Permission
to reproduce any material contained in this work should be obtained from the author.
TABLE OF CONTENTS
LIST OF ABBREVIATIONS ......................................................................................................... 5
PART I: Introduction
GENERAL INTRODUCTION ...................................................................................................... 7
1. TAXONOMY AND HISTORY OF THE GENUS YERSINIA ...................................................................... 8
2. CHARACTERISTICS OF YERSINIA SPP. ......................................................................................... 9
2.1. IN GENERAL ................................................................................................................................ 9
2.2. BIOTYPING AND SEROTYPING ...................................................................................................... 11
2.3. VIRULENCE GENES OF HUMAN PATHOGENIC Y. ENTEROCOLITICA ....................................................... 14
3. EPIDEMIOLOGY: DISTRIBUTION AND TRANSMISSION ROUTE .......................................................... 17
3.1. DISTRIBUTION .......................................................................................................................... 17
3.2. TRANSMISSION ROUTE ............................................................................................................... 19
4. HUMAN YERSINIOSIS .......................................................................................................... 21
4.1. PATHOGENESIS AND SYMPTOMS OF HUMAN INFECTION ................................................................... 21
4.2. INCIDENCE ............................................................................................................................... 24
5. ENTEROPATHOGENIC YERSINIA SPP. IN PIGS ............................................................................. 28
5.1. PRESENCE IN SOWS, BOARS AND PIGLETS....................................................................................... 28
5.2. PRESENCE IN FATTENING PIGS ..................................................................................................... 29
5.3. PRESENCE AND CONTROL ON PIG FARMS ....................................................................................... 35
PART II: EXPERIMENTAL STUDIES
AIMS OF THE THESIS ............................................................................................................ 43
EXPERIMENTAL DESIGN ....................................................................................................... 47
CHAPTER 1: ESTIMATION OF THE WITHIN-BATCH PREVALENCE AND QUANTIFICATION OF
HUMAN PATHOGENIC YERSINIA ENTEROCOLITICA IN PIGS AT SLAUGHTER ............................ 51
CHAPTER 2 : WITHIN-BATCH PREVALENCE AND QUANTIFICATION OF HUMAN PATHOGENIC
YERSINIA ENTEROCOLITICA AND Y. PSEUDOTUBERCULOSIS IN TONSILS OF PIGS AT SLAUGHTER
............................................................................................................................................ 61
CHAPTER 3: SEROPREVALENCE OF ENTEROPATHOGENIC YERSINIA SPP. IN PIG BATCHES AT
SLAUGHTER ......................................................................................................................... 75
CHAPTER 4: PREDICTION OF THE INFECTION STATUS OF PIGS AND PIG BATCHES AT SLAUGHTER
WITH HUMAN PAHTOGENIC YERSINIA SPP. BASED ON SEROLOGICAL DATA .......................... 85
CHAPTER 5: ASSESSMENT OF RISK FACTORS FOR A HIGH WITHIN-BATCH PREVALENCE OF
YERSINIA ENTEROCOLITICA IN PIGS BASED ON MICROBIOLOGICAL ANALYSIS AT SLAUGHTER 99
CHAPTER 6: FACTORS INFLUENCING THE SEROPREVALENCE OF HUMAN PATHOGENIC YERSINIA
SPP. IN FATTENING PIGS AT SLAUGHTER ............................................................................. 117
GENERAL DISCUSSION ........................................................................................................ 127
1. A PREDICTIVE TOOL FOR THE INFECTION STATUS AT SLAUGHTER AGE ............................................. 128
2. THE POSSIBILITIES OF THE FARMER TO INFLUENCE THE PREVALENCE .............................................. 133
3. FINAL IMPACT ON CARCASS CONTAMINATION ......................................................................... 137
4. FUTURE PERSPECTIVES ...................................................................................................... 139
SUMMARY ......................................................................................................................... 141
SAMENVATTING ................................................................................................................ 147
REFERENCES ...................................................................................................................... 153
CURRICULUM VITAE ........................................................................................................... 185
DANKWOORD .................................................................................................................... 195
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LIST OF ABBREVIATIONS Ail attachment-invasion locus
CDE cleaning-disinfection-stand empty
CFU colony forming units
CI confidence interval
CIN cefsulodin-irgasan-novobiocin agar
DNA deoxyribonucleic acid
DWG daily weight gain
EFSA European Food Safety Authority
ELISA enzyme-linked immunosorbent assay
EU European Union
HPI high-pathogenicity island
Inv invasion
KIA kligler iron agar
LPS lipopolysaccharide
MLST multilocus sequence typing
OD optical density
OR odds ratio
PCA plate count agar
PCR polymerase chain reaction
pYV plasmid for Yersinia virulence
RNA ribonucleic acid
Syc specific yop chaperones
T3SS type III secretion system
TSB tryptic soy broth
Ur Urease Broth
VirF virulence factor
VTEC Verotoxigenic Escherichia coli
yadA Yersinia adhesin A
Yop Yersinia outer membrane proteins
YPM Yersinia pseudotuberculosis derived mitogen
Yst Yersinia stable toxin
Ysc Yop secretion
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GENERAL INTRODUCTION
GENERAL INTRODUCTION ________________________________________________________________________
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1. Taxonomy and history of the genus Yersinia
The genus Yersinia is a member of the family of the Enterobacteriaceae, the order of the
Enterobacteriales, the class of the ɣ-proteobacteria and the phylum of the Proteobacteria.
In 1934, only two Yersinia species (at that time Pasteurella) were known, namely
Pasteurella pseudotuberculosis and P. pestis. The first time Y. enterocolitica was
recognized as a new species, was in 1934 by McIver and Pike who called it Flavobacterium
pseudomallei. They found this bacterium in two facial abscesses of a farmer. Five years
later, in 1939, Schleifstein and Coleman proposed the name Bacterium enterocoliticum,
due to new resembling isolates originating from enteric content. The name Yersinia was
first introduced by van Loghem in 1944, who hereby honored the Swiss-born French
bacteriologist Alexandre Yersin, who was the first to describe the plague bacteria, Y.
pestis, in 1894 at the Pasteur Institute in Hong Kong, using the name Pasteurella pestis
(Solomon, 1995; Bottone et al., 1997; Euzéby, 1997; Prentice and Rahalison, 2007). The
name Y. enterocolitica was used the first time by Frederiksen in 1964 and he introduced
the species to the family of the Enterobacteriaceae (Bottone et al., 1997; Bialas et al.,
2012).
Brenner et al. (1976) distinguished the true Y. enterocolitica from the Y. enterocolitica-like
isolates based on DNA relatedness. Thanks to these findings, Bercovier et al. (1980 and
1984), Brenner et al. (1980) and Ursing et al. (1980) identified four Y. enterocolitica-like
species, Y. aldovae, Y. intermedia, Y. kristensenii and Y. frederiksenii. This differentiation
was based on different biochemical reactions (fermentation) of melibiose, rhamnose,
raffinose and sucrose. New species were added overtime. Currently, the genus Yersinia
consists of 18 bacterial species, among which only 3 are pathogenic in humans, i.e., Y.
pestis, Y. pseudotuberculosis and Y. enterocolitica. The remaining 15 species: Y. aldovae,
Y. intermedia, Y. kristensenii, Y. frederiksenii, Y. rohdei, Y. pekkanenii, Y. nurmii, Y.
aleksiciae, Y. similis, Y. bercovieri, Y. mollaretii, Y. philomiragia, Y. ruckeri, Y.
entomophaga, Y. massiliensis are either regarded as avirulent (‘environmental-type’
bacteria) or their plausible pathogenicity has not yet been studied (Euzéby, 1997; Sprague
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and Neubauer 2005; Merhej et al., 2008; Sprague et al. 2008; Hurst et al., 2011; Murros-
Kontiainen , 2011a, 2011b)
2. Characteristics of Yersinia spp.
2.1. In general
Yersinia spp. are facultative anaerobic Gram-negative rods. They are 0.5-0.8 µm in
diameter and 1-3 µm in length. They are nonmotile at 37°C, but motile by peritrichous
flagella when grown below 30°C, except for some Y. ruckeri strains and Y. pestis, which is
always nonmotile. The optimal growth temperature is 28-35°C and can grow on
commonly used media for Enterobacteriaceae (e.g. blood agar, MacConkey agar,
Salmonella-Shigella Deoxycholate Agar). The colonies are lactose-negative. They are
psychrotrophic and able to grow at 4°C, however, this property seems to be more
beneficial for environmental species than for pathogenic ones (Carniel and Mollaret,
1990; Holt et al., 1994).
All three human pathogenic Yersinia spp. target the lymph tissues during infection and
the expression of virulence depends upon the presence of a 70 kb virulence plasmid (pYV),
which is essential for infection of these tissues. Loss of the plasmid leads to avirulence of
the bacteria. This plasmid and its products, the Yops (for Yersinia outer membrane
proteins), are specific for the genus (Carniel and Mollaret, 1990; Wren, 2003, Heesemann
et al., 2006). Yersinia pestis is not a food-born pathogen in comparison with the other two
human pathogenic species that are typical enteropathogens (Biohaz, 2007).
Yersinia pestis is a clone of Y. pseudotuberculosis that diverged 1,500 to 20,000 years ago
by picking up two Y. pestis-specific plasmids, pFra and pPla, which was the key step for
increasing virulence (Achtman et al., 1999; Skurnik et al., 2000; Hinnebusch et al., 2002;
Duan et al., 2014). Yersinia pestis lost non-essential housekeeping genes and has
inactivated certain virulence genes (inv and YadA) encoding for proteins needed for
intestinal pathogenesis (Achtman et al., 1999; Hinnebusch et al., 2002). Rosqvist et al.
(1988) reported a reduced virulence of Y. pestis when activating these inactivated
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virulence genes. The plague microorganism is a non–motile, non–acid, non–sporeforming
cocco–bacillus. When stained with aniline dyes the ends of the bacillus will colour more
intensely, which is known as ‘bipolar staining’. They have a low resistance to
environmental factors: sunlight, high temperatures and desiccation have a destructive
effect, and common disinfectants (e.g. lysol) and preparations containing chlorine kill Y.
pestis within 1 to 10 minutes. The genome consists of a 4.65-Mb chromosome and three
plasmids: pMT1 or pFra (96.2 kb), pYV or pCD (70.3 kb), and the species specific pPla or
pPCP1 (9.6 kb) (Wren, 2003).
The pathogenicity of Y. pseudotuberculosis is associated with several virulence factors
that are encoded on a 70 kb virulence plasmid (pYV) (Cornelis et al., 1998). Additionally,
a chromosomal high-pathogenicity island (HPI) encodes an iron-uptake system
characterized by the siderophore yersiniabactin, the superantigenic toxin Y.
pseudotuberculosis-derived mitogen (YPM) and invasin, which allows an efficient entry
into mammalian cells and plays an important role in systemic infection (Abe et al., 1997;
Fukushima et al., 2001; Grassl et al., 2003; Schubert et al., 2004).
Identification of Y. pseudotuberculosis is challenging because of its indistinguishable
phenotype from the closely related Yersinia similis and Yersinia pekkanenii (Sprague et al.,
2008; Niskanen et al., 2009; Murros-Kontiainen et al., 2011). Yersinia pseudotuberculosis
can be distinguished from Y. similis by 16S rRNA sequencing or by multilocus sequence
typing (MLST) based on housekeeping genes (glnA, thrA, tmk, trpE, adk, argA, aroA) and
from Y. pekkanenii by MLST based on other housekeeping genes (glnA, gyrB, recA and
HSP60) or by DNA-DNA hybridization (Sprague et al., 2008; Laukkanen-Ninios et al., 2011;
Murros-Kontiainen et al., 2011).
Yersinia enterocolitica is pleomorph ranging from small coccobacilli with rounded ends
and bipolar staining to more elongated bacilli. An intriguing feature of Y. enterocolitica is
that the motility is temperature regulated: at 25 °C Y. enterocolitica is peritrichously
flagellated, but at 37 °C they are unflagellated and so nonmotile (Bottone, 1997; Bottone
1999). Based on differences in 16S rRNA and DNA-DNA reassociation values Y.
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enterocolitica has been subdivided into two subspecies: ssp. enterocolitica and ssp.
palearctica (Neubauer et al., 2000a).
Yersinia enterocolitica grows best at a pH between 7.6 and 7.9 and they are highly acid
resistant which is mediated by the ability to produce urease (de Koning-Ward and Robins-
Browne, 1995; Tennant et al., 2008). At low temperature (4°C), the growth is already
inhibited at a pH level that is 0.3-0.5 units higher than the inhibiting pH (on average 4.6)
at 25°C. The optimum growth temperature is set from 32 to 35°C, but even at optimal
conditions for growth, the generation time is quite long (33-39 min at 32°C) compared to
other Enterobacteriaceae (Schiemann, 1980; Adams et al., 1991; Little et al., 1992). They
produce pinpoint colonies after 24 h of incubation, which have a typical bull’s eye
morphology when grown on selective Cefsulodin-Irgasan-Novobiocin (CIN) agar plates
(Schiemann, 1979).
Regarding Y. enterocolitica, there is a high tolerance noticed for surface-active agents like
bile salts and sodium desoxycholate. A high tolerance was also observed for magnesium.
Inhibitors are cetrimide and potassium tellurite. Irgasan was tolerated at concentrations
inhibiting or lethal for other Enterobacteriaceae (Schiemann, 1979; Schiemann, 1980;
Brackett, 1986; Bottone, 1999).
The remaining 15 Yersinia spp. have no reported public health significance. Yersinia
ruckeri is an important fish pathogen causing enteric red mouth disease. It is important in
the aquaculture of rainbow trout in Europe. Symptoms are haemorrhages in various
tissues and organs, particularly around the mouth, in the gills, muscles, peritoneum and
the lower intestine (Carniel and Mollaret, 1990; Huang et al., 2013).
2.2. Biotyping and serotyping
The species Y. pseudotuberculosis is subdivided in 4 biotypes, based on their different
biochemical reaction to raffinose, citrate and melibiose (Fukushima, 2003). Furthermore,
there is a very limited genetic variability between the 21 serotypes, in which Y.
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pseudotuberculosis is currently divided (Palonen et al., 2013). No correlation is found
between a given bioserotype and the severity of the disease which is also depending on
the susceptibility of the host species. Biotyping is infrequently used due to lack of clear
clinical significance (Carniel and Mollaret, 1990). Serotypes O:1a and O:1b are the most
common in Europe, Australia, New Zealand, and North America, and O:4b and O:5b in the
East Asia. Serotype O:6 is only found in Japan. Serotype O:15 strains are prevalent in
human patients in South Korea (Fukushima et al., 2001; Laukkanen-Ninios et al., 2011).
Serotypes O:6 to O:14 have been isolated mainly from animals and environmental sources
(Laukkanen-Ninios et al., 2011).
Yersinia enterocolitica comprises a biochemically heterogeneous collection of organisms.
The species has been divided into six biotypes (1A, 1B, 2-5) based on metabolic
differences, which can be differentiated by biochemical tests (Table I) (Bottone, 1997;
Bottone, 1999). Biotype 1B forms a geographically distinct group of strains that are
frequently isolated in North America (the so-called ‘New-World’ strains) and biotypes 2
to 5 are predominantly isolated in Europe and Japan (‘Old- World’ strains). Biotype 1A is
commonly found in the environment (Tennant et al., 2003). The biotypes can be placed
into three groups: a non-pathogenic group (biotype 1A), which lacks the pYV and seems
to be distantly related to the other biotypes; a weakly pathogenic group that is unable to
kill mice (biotypes 2 to 5); and a group that has significantly higher virulence for mice
(high-pathogenicity group biotype 1B) (Wren, 2003; Schubert et al, 2004; Bhagat and
Virdi, 2007). Yersinia enterocolitica ssp. enterocolitica comprises of biotype 1B and Y.
enterocolitica ssp. paleartica the biotypes 1A and 2-5 (Howard et al., 2006). In whole
genome analysis of 100 Y. enterocolitica strains belonging to different biotypes it was
shown that the biotypes 1A and 1B are more closely related to each other than to other
biotypes, and biotypes 2-5 are very closely related (Reuter et al., 2012).
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Table I. Y. enterocolitica biogrouping scheme (modified from Wauters et al., 1987).
Test Biotype
1A 1B 2 3 4 5
Lipase activity + a + - - - -
Salicin (acid 24 h) + - - - - -
Esculin hydrolysis (24 h) +/- - - - - -
Xylose (acid production) + + + + - V
Trehalose (acid production) + + + + + -
Indole production + + V - - -
Ornithine decarboxylase + + + + + +/(+)
Voges-Proskauer test + + + + + +/(+)
Pyrazinamidase activity + - - - - -
Nitrate reduction + + + + + -
a: + = positive; - = negative; (+) = delayed positive; v = variable.
The species can also be characterized by serotyping (Bottone, 1999). They are
distinguished serologically based on antigenic variation in O-polysaccharides (O-PS; O-
antigen), capsules (K-antigens) and flagellae (H-antigens) (Table II). The O-antigens are the
most important factors responsible for the serological responses. More than 50 serotypes
have by now been distinguished among Y. enterocolitica, of which only 11 serotypes have
been frequently associated with human infection (Bottone, 1999; Hudson et al., 2008;
Virdi and Sachdeva, 2005).
It is well known that certain biotype/serotype combinations are closely correlated with
(1) the geographic origin of the isolates, (2) the ecological niches from which they are
isolated and (3) their pathogenic significance (Bottone, 1999).
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Table II. Pathogenic potential of biotypes and serotypes of Y. enterocolitica (modified from
Bottone, 1999; Biohaz, 2007; Rastawicki et al., 2013).
Biotype Serotypes Virulence
for humans pYV HPIb
Ecologic/geographic
distribution
1A O:8; O:5; O:6,30;
O:7; O:13;O:18;… NP a - -
Environment, pig, food, water, animal and human faeces / global
1B O:7; O:8; O:13;
O:18; O:21;… HP + +
Environment, pig (O:8) / United States, Japan, Europe, The Netherlands (O:8-like), Poland (O:8),
2 O:9; O:5,27 P + -
pig / Europe (O:9), United States (O:5,27), Japan (O:5,27)
3 O:3; O:5,27 P + - Chinchilla (O:1,2,3), pig (O:5,27) / global
4 O:3 P + -
pig / Europe, United States, Japan, South Africa, Scandinavia, Canada
5 O:3; O:2,3;O:1,2,3 P + - hare / Europe
a: NP: Non-Pathogenic; HP: Highly Pathogenic; P: Pathogenic
b: HPI: High Pathogenicity Island
2.3. Virulence genes of human pathogenic Y. enterocolitica
Yersinia enterocolitica possesses both chromosomal and plasmid-associated virulence
determinants. When placed in a medium at 37°C with a low Ca2+ concentration, human
pathogenic Y. enterocolitica is producing and secreting certain proteins (Cornelis, 1998).
To express the full potential virulence, human pathogenic Y. enterocolitica needs a
plasmid for Yersinia virulence (pYV) encoding approximately 50 proteins (Zink et al., 1980;
Portnoy et al., 1981).
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A first group of virulence determinants are the adhesins. The chromosomally encoded
Invasin (Inv) is a polypeptide of which synthesis in Y. enterocolitica is growth phase-
dependent. It is maximally expressed when bacteria transit from logarithmic to stationary
phase of growth at both 28 and 37°C (Bottone, 1999; Pepe et al., 1994). Inv is directly
involved in the first phase of infection and initiates the internalization of the bacteria in
small intestine epithelial cells, especially to M cells by binding to β1 integrines (Isberg,
1990; Jepson and Clark, 1998). It stimulates the remodeling of actin filaments in the M-
cell cytoskeleton, assists the induction of autophagocytosis in macrophages and initiates
the cytokine production (Grassl et al., 2003; Deuretzbacher et al. 2009). The chromosomal
attachment-invasion locus (Ail), is exclusively produced at 37°C (Miller and Falkow, 1988;
Pederson and Pierson, 1995). It supports the adhesion to epithelial cells and it binds to
laminin and fibronectin (Miller et al., 2001; Mikula et al., 2013). The third outer membrane
adhesin encoded by the chromosome, pH6, supports the resistance to phagocytosis,
assists the haemagglutination and tissue adhesion (Chen et al., 2006). The Yersinia
adhesin (yadA) gene is located on the pYV and its expression is mainly temperature-
regulated. The proteins are only produced at 37°C. YadA assists the adhesion to epithelial
cells, neutrophils, and macrophages, the binding to collagen, fibronectin, and laminin, the
invasion of epithelial cells and the Yop (Yersinia outer membrane proteins) delivery
(Heesemann et al., 1987; Visser et al., 1995; Mikula et al., 2013). It also protects the
bacteria against phagocytosis of polymorphonuclear leukocytes and monocytes and
initiates the down regulation of Inv (Mikula et al., 2013). The two regulators of the yadA
gene transcription are VirF (plasmid-borne transcriptional activator of yadA) and YmoA
(chromosome encoded transcriptional repressor of virF). The YmoA protein also inhibits
the expression of the inv gene and the enterotoxin YstA gene (Yersinia stable toxins),
participates in the temperature-dependent synthesis and secretion of the Yops, and the
production of YadA and VirF (Lambert de Rouvroit et al., 1992; Platt-Samoraj et al., 2006;
Bancerz-Kisiel et al., 2012). YstB is correlated with Y. enterocolitica biotype 1A
(Ramamurthy et al., 1997; Bancerz-Kisiel et al., 2012). Lack of pathogenicity in 1A biotype
strains is currently under debate, due to the appearance of production of this toxin
(Bancerz-Kisiel et al., 2012).
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The second virulence determinants, the lipopolysaccharide (LPS) is found on the outer
membrane of Gram-negative bacteria. One LPS molecule exists of three parts: 1) lipid A
that is anchored in the outer membrane and is an endotoxin, 2) the core oligosaccharide
with its internal and external parts and 3) the O-specific polysaccharide that has antigenic
properties and is exposed to cell surrounding. The LPS core functions as a bacteriophage
receptor, activates the host complement system and interacts with serum proteins other
than antibodies (Skurnik et al., 1999; Najdenski et al., 2003; Biedzka-Sarek et al., 2008;
Pinta et al., 2009).
The last group of virulence determinants is a set of at least 12 proteins called Yops. Most
of the yop genes have been identified and sequenced, and they appeared to be almost
identical in the three human pathogenic Yersinia spp. Although initially described as outer
membrane proteins, the Yops were also recovered from the culture supernatant, and it
was later found that they were actually secreted proteins (Michiels et al., 1990). The Yop
virulon consists of two groups of Yops: some are intracellular effectors (effector Yops)
delivered inside eukaryotic cells, while others form an extracellular delivery apparatus
which is necessary for injecting the effectors across the plasma membrane of eukaryotic
cells (translocator Yops) (Cornelis et al., 1998; Cornelis, 2002). The secretion of Yops
requires a specific protein pump (Ysc: ‘Yop secretion’), which is part of a type III secretion
system (T3SS) and is also encoded by the pYV (Michiels et al., 1991). The secretion
requires a complex machinery made of at least 28 proteins. To protect the Yops against
degradation before attachment to a cell surface or secretion, chaperones (Syc: ‘specific
yop chaperones’) are produced (Wattiau and Cornelis, 1993; Wattiau and Cornelis, 1994;
Wattiau et al., 1994; Cornelis et al., 1998; Cornelis, 2002).
Until now, 6 effector proteins (YopE, YopH, YopO, YopM, YopP, and YopT) are known to
be translocated through the eukaryotic membrane. The injected Yops support the survival
of the invading bacteria, by disturbing the dynamics of the cytoskeleton, disrupting
phagocytosis, and blocking the production of pro-inflammatory cytokines (Rosqvist et al.,
1994; Persson et al., 1995; Boland et al., 1998; Cornelis et al., 1998; Iriarte and Cornelis,
1998; Cornelis, 2002). YopD assists with the delivery of YopE, while YopB helps deliver
YopE and YopH (YopD and YopB are translocator Yops). About 50 genes are involved in
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the T3SS, occupying 75% of the pYV (Cornelis, 1998). The production of Yops can be
initiated in vitro by a low Ca2+ concentration (Gemski et al., 1980).
At last, Y. enterocolitica 1B have a chromosomally encoded 35 – 45 kb high pathogenicity
island (HPI) encoding genes involved in yersiniabactin-mediated iron uptake (Pelludat et
al., 1998). This biotype also has yts1 type II and ysa type III secretion systems which
enhance virulence (Haller et al., 2000; Iwobi et al., 2003).
3. Epidemiology: distribution and transmission route
3.1. Distribution
Yersinia pestis
Yersinia pestis caused three pandemics, each started by a different biovar: Antiqua
(Mediterranean Sea – 600 AC), Medievalis (Europe – 14th century) and Orientalis (China –
middle 19th century). Biovar Medievalis, also called the Black Death, decimated the
population in Europe (Stenseth et al., 2008). Biovar Orientalis caused outbreaks of plague
in Asia until the beginning of the 20th century. The three pandemics killed more than 200
million people. Today it is believed to exist no longer in Europe or in Australia (Schubert
et al., 2004; Stenseth et al., 2008; EFSA and ECDC, 2013).
The pathogen circulates in animal reservoirs, particularly in rodents. They are the main
source of Y. pestis but they are also sensitive to it. The natural foci are situated in a broad
belt in the tropical and sub–tropical latitudes. However, within this belt, many areas are
free of the plague (e.g. the desert and large areas of continuous forest) (Schubert et al.,
2004).
Yersinia pseudotuberculosis
Yersinia pseudotuberculosis is an important causal agent of zoonosis with global
distribution (Fukushima et al., 2001). Y. pseudotuberculosis is able to survive for long
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periods in soil and water (river and fresh water), and the contamination of food and water
can be a potential source of infection (Fukushima, 1992; Fukushima et al., 1995; Han et
al., 2003). The most common reservoirs for Y. pseudotuberculosis have been reported to
be carrots and lettuce (Jalava et al., 2006). The pathogen has been recovered from diverse
animal sources ranging from farm animals, pets and wild animals. It was found in wild
mammals like bats, raccoon dogs, deer, hares, rabbits, mouflons, buffaloes, boars and
foxes, in birds like ducks and in small rodents (Jerrett et al., 1990; Riet-Correa et al., 1990;
Fukushima and Gomyoda, 1991; Nikolova et al., 2001; Backhans et al., 2011; Fredriksson-
Ahomaa et al., 2011; Nakamura et al., 2013). Farm animals (pigs, cattle, sheep and goats)
are also possible carriers (Philbey et al., 1991; Lanada et al., 2005; Hodges and Carman,
2011; Martinez et al., 2011; Novoslavskij et al., 2013). More information about the
presence in pigs and pig batches is given in ‘5. Enteropathogenic Yersinia spp. in pigs’.
Human pathogenic Y. enterocolitica
The consumption of pork is the main source for human infection and healthy pigs are
known to be the primary reservoir of the human pathogenic types of Y. enterocolitica,
mainly biotype 4 (serotype O:3) (Tauxe et al., 1987; Ostroff et al., 1994; Bottone, 1999;
Fredriksson-Ahomaa et al., 2006; Fosse et al., 2009; Huovinen et al., 2010; EFSA and ECDC,
2013; Rosner et al., 2012). For an exhaustive overview about the presence in pigs and on
pig farms, see ‘5. Enteropathogenic Yersinia spp. in pigs’. Other food producing animals
seldom carry bioserotype 4/O:3. Milnes et al. (2008) found 3.0% of sheep positive for
human pathogenic Y. enterocolitica biotype 3 (O:4,32, O:5 and O:5,27). McNally et al.
(2004) also found bioserotype 3/O:5.27 in 35% of the sheep and 4% of the cattle. Goats
can harbor biotypes 2, 3 and 5 (Philbey et al., 1991; Nikolova et al., 2001; Lanada et al.,
2005; Milnes et al., 2008; EFSA and ECDC, 2013). High prevalence of anti-YOP antibodies
in goats (66%) and sheep (56%) in Northern Germany has been reported (Nikolaou et al.,
2005). Chickens can harbor serotypes non O:3 and non O:9, which is not confirmed to be
pathogenic (Kechagia et al., 2007).
Companion animals, like dogs and cats, can carry bioserotype 4/O:3 (Fredriksson-Ahomaa
et al., 2001c; Bucher et al., 2008). They possibly receive their Y. enterocolitica 4/O:3 from
contaminated pork (Fredriksson-Ahomaa et al., 2001c).Wild animals, like rabbits, boars,
GENERAL INTRODUCTION ________________________________________________________________________
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19
Asiatic jackals, red foxes, ibexes and wild cats are also hosts of Y. enterocolitica, mostly
serotype O:3 (Nikolova et al., 2001; Al Dahouk et al., 2005; Fredriksson-Ahomaa et al.,
2009a; Wacheck et al., 2010; Joutsen et al., 2013). Even birds can be carriers (Niskanen et
al., 2003). At last, small rodents can carry bioserotype 4/O:3 (Pocock et al., 2001; Bucher
et al., 2008; Backans et al., 2011). Clinical disease in animal reservoirs is uncommon
(Bottone, 1999). Barre et al. (1976) isolated human pathogenic Y. enterocolitica serotypes
O:10, O:10,14,16 and O:4,16 from soil samples.
3.2. Transmission route
In general
Yersinia pestis is transmitted by fleas, while the other two species of human pathogenic
Yersinia are typically transmitted orally. They both can be transmitted by the consumption
of contaminated food (most important, see section ‘3.2.2. Presence of enteropathogenic
Yersinia spp in food’), untreated, contaminated water, direct contact with infected
animals or even person-to-person transmission involving faecal-oral contamination
(Gutman et al., 1973; Toivanen et al., 1973; Tauxe et al., 1987; Fukushima et al., 1988;
Carniel and Mollaret, 1990; Han et al., 2003; Kangas et al., 2008; Stenseth et al., 2008;
Rimhanen-Finne et al., 2009; EFSA and ECDC, 2013). Fredriksson-Ahomaa et al. (2006)
found indistinguishable genotypes between strains from humans and strains from dogs,
cats, sheep and wild rodents, indicating that these animals are a possible source for
human infections. It is reported that children can get infected by cat-contaminated
environmental substances (Fukushima et al., 1989a). At last, blood transfusion is also a
possible infection route. Blood transfusion with Y. enterocolitica infected blood resulted
in 70% of the cases in the death of the receiver. The few bacteria present in the stored
blood multiply at 4°C and produce a septic shock shortly after transfusion. The first case
of infected blood transfusion ever reported was in 1975. A 57-year-old woman from the
Netherlands got a septic shock one hour post transfusion but survived (Jacobs et al., 1989;
Mollaret et al., 1989; Guinet et al., 2011).
GENERAL INTRODUCTION ________________________________________________________________________
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Presence of enteropathogenic Yersinia spp. in food
A great variety of food can be contaminated with enteropathogenic Yersinia spp.
Raw pork and products derived from pork are the most important source of infection:
strains isolated from humans have genotypes indistinguishable from the genotypes found
in pigs and pork and in case control studies, human infection has been associated with the
consumption of pork (products) (Fredriksson-Ahomaa et al., 2001b; Jones et al., 2003;
Fearnley et al., 2005; Fredriksson-Ahomaa et al., 2006; Grahek-Ogden et al., 2007; Boqvist
et al., 2009; Huovinen et al., 2010). Pig tonsils and intestines are often infected, which can
lead to contamination of the carcass during slaughter (Nesbakken, 1985; Nesbakken,
2000; Fredriksson-Ahomaa et al., 2001a; Simonova et al., 2008; Laukkanen et al., 2009;
Van Damme et al., 2013). The prevalence on pork carcasses varies between 0 and 63%
(Nesbakken, 1988; Fredriksson-Ahomaa et al., 2000b; Boyapalle et al., 2001; Nesbakken
et al., 2003; Gürtler et al., 2005; Lindblad et al., 2007; Nesbakken et al., 2008; Wehebrink
et al., 2008; Bonardi et al., 2013; Van Damme et al., 2013). Furthermore, human
pathogenic Y. enterocolitica have frequently been isolated from pork products and edible
offals (Fredriksson-Ahomaa et al., 2007b; Bucher et al., 2008; Hudson et al., 2008; Bonardi
et al., 2010; Messelhäusser et al., 2011, Tan et al., 2014). The most contaminated pork
products are pig tongues, with an occurrence of 11 to 98%. In minced pork, 0-32% of the
samples are contaminated with human pathogenic Y. enterocolitica (Wauters et al., 1988;
Kwaga et al., 1990; Fredriksson-Ahomaa et al., 1999; Boyapalle et al., 2001; Vishnubhatla
et al., 2001; Bucher et al., 2008; Messelhäusser et al., 2011; Tan et al., 2014). Pathogenic
Y. enterocolitica has been isolated from the worktable and the metal glove in a butcher
shop, enabling cross-contamination at retail level (Fredriksson-Ahomaa et al., 2004).
Human pathogenic Y. enterocolitica were also detected in meat products, originating from
non-porcine animals. In 2011, there was one sample reported of bovine origin containing
Y. enterocolitica (EFSA and ECDC, 2013). Tan et al. (2014) detected by PCR 4/6 raw beef
samples as positive. Bucher et al. (2008) did not find contaminated raw beef. Yersinia
enterocolitica was also demonstrated in meat from goats, sheep, horses, donkeys, bison
and water buffalos, as well as in fish (EFSA and ECDC, 2013). Messelhäusser et al. (2011)
found three PCR-positive samples out of 51 game meat samples. Also raw poultry samples
GENERAL INTRODUCTION ________________________________________________________________________
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21
can be contaminated, even after they have been frozen (Norberg, 1981; Bonardi et al.,
2010; Tan et al., 2014). Even on raw seafood, Y. enterocolitica can be found (Tan et al.,
2014). At last, contamination of eggs is also possible (Favier et al., 2005).
Milk and milk products (e.g. chocolate milk) can also be contaminated with Y.
enterocolitica, however in the EU there were no contaminated milk samples reported in
2011 (Black et al., 1978; Schiemann and Toma, 1978; Jayarao and Henning, 2001; EFSA
and ECDC, 2013). By using qPCR, it was possible to detect and quantify ail positive raw
cow milk samples in Belgium (Najdenski et al., 2012) in contrast to Messelhäusser et al.
(2011) in Germany. On the other hand, the bacteria were already isolated from
pasteurized milk. Since Y. enterocolitica does not survive the pasteurization processes of
dairy products, the presence of these pathogens in pasteurized milk is probably the result
from process failure or recontamination after pasteurization (Lovett et al., 1982; Walker
and Gilmour, 1986).
Sometimes, enteropathogenic Y. enterocolitica can be found on vegetables (Lee et al.,
2004). Tan et al. (2014) did not found contaminated samples. In 2011, there were no
positive samples found in the European Union (EU) (EFSA and ECDC, 2013).
4. Human yersiniosis
4.1. Pathogenesis and symptoms of human infection
Yersinia pestis
The life cycle of Y. pestis is completely distinct from that of enteropathogenic Yersiniae.
The plague primarily affects wild rodents. It spreads between rodents and to other
animals via fleas (most common), cannibalism or (possibly) contaminated soil. The disease
spreads among the human population via bites of contaminated fleas and causes bubonic
plague, or via aerosols produced by coughing of people with the pneumonic plague.
Humans bitten by an infected flea usually develop a bubonic form of plague, which is
GENERAL INTRODUCTION ________________________________________________________________________
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22
characterized by a swelling of the lymph node draining the flea bite site. Initial symptoms
of bubonic plague appear 7–10 days after infection. If diagnosed early, bubonic plague
can be successfully treated with antibiotics. Most of the time, the bacteria invade the
bloodstream and spread to the whole body where they localize mainly in the spleen, liver
and lungs. The septicemia is responsible for the fatal outcome of bubonic plague. The
mortality rate depends on how soon treatment is started, but is always very high (40%–
70% mortality). If the bacteria reach the lungs, the patient develops pneumonia
(pneumonic plague). Pneumonic plague is one of the most deadly infectious diseases:
patients can die 24 h after infection. During this period, the infected patient is highly
infectious. Y. pestis is considered one of the most pathogenic bacteria for humans (Carniel
and Mollaret, 1990; Stenseth et al., 2008).
Yersinia pseudotuberculosis and Y. enterocolitica
After oral uptake of the enteropathogenic Yersinia spp., they attach to the intestinal brush
border of the terminal ileum and proximal colon. This is also the location of the Peyer's
Patches (PP), the preferred place to invade the body and which are covered by M-cells
(specialized cells of antigen uptake). Yersinia enterocolitica and Y. pseudotuberculosis
penetration of M-cells is mediated by three invasion genes (inv, ail, and yadA). Bacterial
surface structures produced at lower temperatures are already covering the bacterial cell
surface before uptake (Pepe and Miller, 1993). YadA mediates mucus and epithelial cell
attachment, Inv directly promotes early epithelial cell penetration by attaching to β1
integrins on eukaryotic cell surfaces (Isberg, 1990). Ail, only produced at body
temperature, also enhances epithelial cell penetration (Miller and Falkow, 1988; Isberg,
1990). The remodeling of actin filaments in the M-cell induced by Inv is the start of the
invagination of the bacteria into the cells, ending within endosomal vesicles. These
vesicles transport the bacteria through the M-cell and release them into the lamina
propria where they stay from now on mostly extracellular (Autenrieth and Firsching, 1996;
Grassl et al., 2003). After this translocation, Inv binds to the β1 integrins, which induces
the chemokine production (e.g. IL-8). In the PP, after host cell adhesion, the Ysc is
established and initiates the cellular killing. YopH dephosphorylates eucaryotic proteins,
especially in phagocytic cells, which interferes with the signal transduction pathways of
the target cell and thereby impedes phagocytosis. YopB suppresses the production of the
GENERAL INTRODUCTION ________________________________________________________________________
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23
tumor necrosis factor alpha (TNF-α). Further, to enhance a systemic spread, resistance to
complement-mediated has been correlated with the presence of two outer membrane
proteins, YadA and Ail (Martinez, 1989). The Yersinias replicate and express YadA, which,
as described above, protects against the phagocytosis of recruited polymorphonuclear
leukocytes and monocytes (Mikula et al., 2013). Three days post infection, the whole PP
is colonized and its normal architecture is destroyed. Moreover, the bacteria create gaps
into the basal lamina of the M-cells through which they could freely pass into the lamina
propria (Bottone 1997).
There are no striking differences between the enteric symptoms caused by Y.
enterocolitica or Y. pseudotuberculosis. Yersinia pseudotuberculosis infections are more
common in adults than those caused by Y. enterocolitica which occur mostly in young
children. The symptoms for a Y. pseudotuberculosis infection are more severe than the
symptoms caused by Y. enterocolitica. Both can present right-sided abdominal pain
simulating appendicitis and fever, which is more likely in older children and adults for Y.
enterocolitica. Yersinia pseudotuberculosis is clinically manifested as enteritis, mesenteric
lymphadenitis and occasionally septicemia. Symptoms of infections with Y. enterocolitica
are nausea, diarrhea, sometimes bloody, and in elderly persons and in patients with
underlying conditions (iron overload, cirrhosis, diabetes, cancer,...), systemic forms of the
disease are often observed. Symptoms typically develop four to seven days after exposure
and last on average one to three weeks. The disease is self-limited and rarely lethal for
humans (Cover and Aber, 1989; Bottone, 1997; Sakai et al., 2005; EFSA and ECDC, 2013).
Due to YadA, its ability to adhere to collagen and the resulting immunologic reaction, Y.
enterocolitica can cause reactive arthritis (a sterile, immunomediated inflammation of the
joints) and erythema nodosum, which are the two most common sequelae of this
infection (Cover and Aber, 1989; Rosner et al., 2013). Reactive arthritis is mostly seen in
adults, with a higher incidence in patients who are human leucocyte antigen (HLA)-B27-
positive. It develops 1-2 weeks post infection, and lasts for several months or years
(Hannu et al., 2003). Other symptoms such as pneumonia, pharyngitis, encephalitis,
septicemia and formation of abscesses in liver and spleen may occur (Pulvirenti et al.,
2007; Stolzel et al., 2009; Ito et al., 2012; Wong et al., 2013).
GENERAL INTRODUCTION ________________________________________________________________________
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24
4.2. Incidence
The last known figures are those from 2011, since the last joint report of EFSA and ECDC
does not mention the incidence of Yersinia spp. (EFSA and ECDC, 2013; 2014). In 2011,
there were 7,017 confirmed cases of yersiniosis reported in the EU. The number of cases
increased by 3.5 % compared to 2010 (n = 6,780), which was the first time a slight increase
was observed since 2006. Yersiniosis was the fourth most frequently reported zoonosis in
the EU, after Campylobacter, Salmonella and Verotoxigenic Escherichia coli (VTEC). The
notification rate in Europe was 1.63 cases per 100,000 inhabitants in 2011, which was very
similar in the United States, where it was set at 1.6 (EFSA and ECDC, 2013; Scallan et al.,
2013). In Belgium, there were 206 cases registered by the Sentinel Laboratory Network in
2011 (FAVV-AFSCA, 2012). Nevertheless, many infections are not confirmed, reported or
even not detected. It is estimated that for each infection with Y. enterocolitica, there are
48 infections not detected (Scallan et al., 2013). Up to 40% of the German population has
antibodies against Yersinia (Neubauer et al., 2001).
In Europe, there is a predominance of human pathogenic Y. enterocolitica biotype 4
(serotype O:3) and, less commonly, biotype 2 (serotype O:9, O:5,27). Species information
was available for 6,830 of 7,017 confirmed cases: 98.4% were Y. enterocolitica, followed
by Y. pseudotuberculosis (0.9%) and other species (0.6%) (EFSA and ECDC, 2013).
The highest country-specific notification rates were observed in Lithuania and Finland,
respectively 11.40 and 10.31 cases per 100,000 inhabitants. In 2011, from 773 confirmed
yersiniosis cases, 427 (55%) were hospitalized. Most of them (258 cases, 60 %) were
observed in Lithuania. The highest proportion of hospitalized cases by country was
reported in Romania (80.9 %) (EFSA and ECDC, 2013). In Ireland, there are very few human
cases reported per year, however, based on serology testing, in 25% of the sampled
people presence of YOP-antibodies was demonstrated (Ringwood et al., 2012). In the
United States, it is also a relatively uncommon cause of sporadic disease, accounting for
<0.3% of all foodborne illness (Mead et al., 1999).
GENERAL INTRODUCTION ________________________________________________________________________
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The reported outbreaks worldwide of human pathogenic Y. enterocolitica and Y.
pseudotuberculosis occurring this century are listed in Table III. Infection mostly occurs in
young children (Jones et al., 2003; Sakai et al., 2005; Moriki et al., 2010). While reports of
food borne outbreaks caused by Y. pseudotuberculosis are rare worldwide, several
outbreaks have been detected in Finland and were caused by the consumption of fresh
vegetables (Jalava et al., 2006; Kangas et al., 2008; Rimhamen-Finne et al., 2009).
Table III: Literature reported outbreaks of human pathogenic Y. enterocolitica and Y. pseudotuberculosis since 2000.
Bio/ serotype
Period Patients Source of infection
Symptoms (% of patients showing symptoms)
Place Reference
number Range age (median)
Location Country
Human pathogenic Y. enterocolitica NSa November
2001 12 0.1-0.7 (0) chitterlings - diarrhea (100)
- bloody stools (70) - vomiting (70) - fever (80)
households U. S. Jones et al., 2003
O:3 January 2002
22 30-60 (NS) unknown - fever (40.9) - abdominal pain
(45.4) - diarrhea (13.6)
oil tanker Croatia Babic-Erceg et al., 2003
O:8 August 2004
42 <6 (NS) salad - fever (100) - abdominal pain
(56) - diarrhea (37) - vomiting (12)
school Japan Sakai et al., 2005
O:3 2005 6 NS raw milk - NS NS Austria Much et al., 2007
O:9 December 2005
11
10-88 (44) ready-to-eat pork product
- severe abdominal pain
- diarrhea - fever - joint pain - vomiting
households Norway Grahek-Ogden et al., 2007
2/O:9 July 2006
3 1-68 (5) pork - enterocolitis - bloody stool - diarrhea - fever
one household Japan Moriki et al., 2010
Bio/ serotype
Period Patients Source of infection
Symptoms (% of patients showing symptoms)
Place Reference
number Range age (median)
Location Country
O:9 January 2011
21 10-63 (30-39)
ready-to-eat salad mix
- gastroenteritis nationally Norway MacDonald et al., 2011
Y. pseudotuberculosis O:1 May
2003 111 4-52 (10) carrots - abdominal illness
(53) - erythema
nodosum (48) - reactive arthritis
(1.5)
school, day-care
Finland Jalava et al., 2006
O:1 March 2004
53 7-18 (NS) carrots - gastroenteritis school
Finland Kangas et al., 2008
O:1 August 2006
427 12-60 (15) carrots - fever (95) - acute abdominal
pain (97) - back pain (40) - joint pain (38) - diarrhoea (20) - erythema
nodosum (15) - vomiting (14)
school, day-care
Finland Rimhamen-Finne et al., 2009
a: NS: Not specified
GENERAL INTRODUCTION ________________________________________________________________________
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28
5. Enteropathogenic Yersinia spp. in pigs
Pigs are asymptomatic carriers of human pathogenic Y. enterocolitica and Y.
pseudotuberculosis in the tonsils and the intestines (Nesbakken, 1985; Bottone, 1999;
Nesbakken, 2000; Fredriksson-Ahomaa et al., 2001a; Simonova et al., 2008). They do not
develop serious illness because piglets are capable of restricting colonization by Y.
enterocolitica to the throat and the intestines (Schiemann, 1988).
5.1. Presence in sows, boars and piglets
Pathogenic Y. enterocolitica and Y. pseudotuberculosis are detected at a significantly
lower rate in sows than in fattening pigs. Previous studies show maximum 14% sows
infected with Y. enterocolitica, while no sows were found positive for Y.
pseudotuberculosis (Niskanen et al., 2002; Korte et al., 2004; Gürtler et al., 2005; Bowman
et al. 2007; Wehebrink et al., 2008; Farzan et al., 2010). Bowman et al. (2007) found that
2.4% of the gestating sows were positive for ail-positive Y. enterocolitica, while the
pathogen was never detected in sows in the farrowing unit. Some studies suggest that
older sows may develop a natural resistance to enteropathogenic Yersinia spp.
(Fukushima et al., 1984a; Niskanen et al., 2002; Korte et al., 2004; Nesbakken et al., 2006).
Niskanen et al. (2008) has performed the only reported study on the prevalence in boars.
Six boars were studied, none of them were positive.
The prevalence in piglets rose to 3% (Gürtler et al., 2005; Bowman et al. 2007; Wehebrink
et al., 2008; Farzan et al., 2009). Bowman et al. (2007) suggest that there is a trend of
increasing prevalence as piglets get older. They found 0.5% of the suckling piglets and
0.6% of the nursery pigs positive for ail-positive Y. enterocolitica. Nevertheless, Gürtler et
al. (2005) observed no positive suckling piglets at an age of 3 days or 3 weeks, or at an age
of 10 weeks (nursery unit).
GENERAL INTRODUCTION ________________________________________________________________________
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29
5.2. Presence in fattening pigs
The occurrence of these bacteria in fattening pigs is depending on the age. Bowman et al.
(2007) found more Y. enterocolitica positive pigs in the late fattening stage compared to
the early fattening stage. Gürtler et al. (2005) obtained similar results, with 2.8% of 14-
week old pigs positive and 19.6% of the 20-week old fattening pigs harbored human
pathogenic Y. enterocolitica.
Two methods can be used to define the prevalence in pigs: a microbiological and a
serological way. Serological analysis is based on the antigenic properties of the discussed
virulence factors LPS and Yops which are both important for the production of antibodies.
These antibodies can be detected by using an enzyme-linked immunosorbent assay
(ELISA). An LPS-ELISA only detects antibodies against certain serotypes of which the
antigen is included in the ELISA (Thibodeau et al., 2001). An ELISA based on Yops detects
all Yersinia spp. containing the pYV (Y. pestis, Y. pseudotuberculosis and human
pathogenic Y. enterocolitica) (Labor Diagnostik Leipzig, Qiagen, Leipzig, Germany).
There are time-dependent differences in analyzing tonsils, faeces or meat juice/blood
(Fig. I).
Fig. I. The occurrence of Y. enterocolitica O:3 in tonsils and faeces and of antibodies
against Y. enterocolitica O:3 in blood of 60 animals in relation to age (modified from
Nesbakken et al., 2006).
0
20
40
60
80
100
60 to 65 86 to 93 102 to 107 133 to 135 147 to 162
pro
port
ion o
f posi
tive
anim
als
for
Y. en
tero
coli
tica
(%
)
days of age
tonsils
faeces
antibodies
GENERAL INTRODUCTION ________________________________________________________________________
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30
Some experimental studies about the evolution of antibodies and bacteriology of infected
pigs were performed. Nielsen et al. (1996) inoculated 25 pigs with Y. enterocolitica O:3.
These pigs had culture-positive faeces from day 5 to 21 post infection (p.i.), where after
shedding of Yersinia declined to <10% of the pigs at day 49 p.i. and to 0% at day 68 p.i.
Using an indirect pig LPS-ELISA, sera from all pigs showed an increase of antibody titer. All
inoculated pigs had seroconverted at day 19 p.i. and remained seropositive until day 70
p.i. with a maximum level at day 33 p.i. Nesbakken et al. (2006) studied the natural
dynamic of infection: between 100 and 180 days of age, serology (LPS-ELISA) could be
used to differentiate between infected and non-infected pigs. Bacteriological examination
of faeces could be used for the same purpose between 85 and 135 days of age, while
bacteriological examination of tonsils could be applied from 85 to 180 days. Vilar et al.
(2013) showed a peak in Yersinia-excretion in pigs of 2-3 months old, but the antibody-
titer was rising till 5 months. Other studies performed on the natural dynamics of infection
also show more excreting pigs of 12-21 weeks old, followed by a decrease (Fukushima et
al., 1983; Gurtler et al., 2005; Virtanen et al., 2012). Infection can be detected earlier by
using the microbiological method instead of serology (Fukushima et al., 1983; Nesbakken
et al., 2006; Nielsen et al., 1996). The dilemma of analyzing tonsils or faeces is depending
on the time of infection. The carriage of enteropathogenic Y. enterocolitica lasts several
months in the tonsils, whereas faecal excretion decreases within a few weeks p.i.
(Fukushima et al., 1983; Fukushima et al., 1984a; Fukushima et al., 1984b; Nesbakken et
al., 2006; Nielsen et al., 1996; Virtanen et al., 2012). Besides, intestinal colonization
continues for a long time and does not occur by re-infection (Fukushima et al., 1983). The
difference between tonsillar and faecal sampling is not striking for Y. pseudotuberculosis
(Laukkanen et al., 2008).
There are already many studies performed about the presence of these bacteria in
fattening pigs at slaughter in different countries and ranges, based on isolation of tonsils,
from 2 to 93%, and based on isolation of faeces, from 4 to 30% (Tables IV and V). Mostly,
Y. enterocolitica bioserotype 4/O:3 was isolated. The number of studies based on serology
is nevertheless very limited. Nesbakken et al. (2006) sampled two farms overtime, 20-
100% of the fattening pigs showed antibodies against Y. enterocolitica O:3 by LPS-ELISA.
GENERAL INTRODUCTION ________________________________________________________________________
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31
Thibodeau et al. (2001) found an overall prevalence of 66% in 291 tested animals, using
an LPS-ELISA. In the study of von Altrock et al. (2011) 80 batches (30 pigs/batch) were
tested, with an overall prevalence of 64.1% using an ELISA based on Yops.
Table IV. The reported presence of Y. pseudotuberculosis in fattening pigs at slaughter.
Country Number of farms
Number of pigs
Positive batches
(%)
Number of positive pigs
Bioserotypes Reference Infected tonsils
(%)
Infected faeces
(%)
Belgium 10 201 NSa 5 (2) - 1/O:1 (1), 1/O:2 (1), 2/O:3 (3) Martinez et al., 2011 China NS 4495 NS 4 (0.1) - NS Liang et al., 2012
NS 3039 NS - 0 NS Liang et al., 2012 Estonia 15 151 2 (13) 2 (1) - 2/O:3 Martinez et al., 2009 Finland 55 301 NS 8 (3) 13 (4) O:3 Laukkanen et al., 2010b
15 350 6 (40) 34 (10) - O:3 Laukkanen et al., 2008 15 358 4 (27) - 24 (7) O:3 Laukkanen et al., 2008 NS 210 NS 8 (4) - 2/O:3 Niskanen et al., 2002
Germany NS 631 NS - 5 (1) O:2 (3), O:3 (2) Weber and Lembke, 1981 Great Britain 45 630 35 (78) 114 (18) - 1/O:1 (32), 1/O:4 (29), 2/O:3 (41) Martinez et al., 2010
Greece NS 455 NS 3 (0.7) - NS Kechagia et al., 2007 Italy 22 428 3 (14) 5 (1) - 1/O:1 (3), 2/O:3 (1), 2/NTb (1) Martinez et al., 2011
25 98 1 (4) - 1 (1) NS Bonardi et al., 2007 Japan 96 1200 15 (16) - 33 (3) O:1 (7), O:2 (3), O:3 (16), O:4b (6) Fukushima et al., 1989b Latvia 47 404 6 (13) 12 (3) - NS Terentjeva and Berzins, 2010
5 109 3 (60) 5 (5) - 2/O:3 Martinez et al., 2009 Lithuania 11 110 6 (55) - 11 (10) 2/O:3 Novoslavskij et al., 2013
Russiac 10 197 6 (60) 13 (7) - 2/O:3 Martinez et al., 2009
aNS: not specified bNT: not typable cRussia: Leningrad Region
Table V. The reported presence of human pathogenic Y. enterocolitica in fattening pigs at slaughter by isolation.
Country Number of farms
Number of pigs
Positive batches
(%)
Number of positive pigs
Bioserotypes Reference Positive tonsils
(%)
Positive faeces
(%)
Belgium 10 201 8 (80) 89 (44) - 3/O:9 (8), 4/O:3 (81) Martinez et al., 2011 - 139 - 52 (37) - 4/O:3 Van Damme et al., 2010
Canada 264 395 NSa 7 (2) - 2/O:5,27 (2), 4/O:3 (5) O’Sullivan et al., 2011 China - 4495 - 694 (15) - NS Liang et al., 2012
- 1239 - - 70 (6) NS Liang et al., 2012 Denmark - 195 - 164 (84) - 4/O:3 Rasmussen et al., 1995 Estonia 15 151 15 (100) 135 (89) - 4/O:3 Martinez et al., 2009 Finland 55 301 NS 177 (59) 90 (30) 4/O:3 Laukkanen et al., 2010b
- 204 - - 68 (33) 3/O:3 (1); 4/O:3 (67) Laukkanen et al., 2010a 15 350 12 (80) 124 (35) - 4/O:3 Laukkanen et al., 2008 - 210 - 109 (52) - 4/O:3 Korte et al., 2004 - 185 - 48 (26) - 4/O:3 Fredriksson-Ahomaa et al., 2000a
Germany - 164 - 101 (62) 17 (10) 4/O:3 Bucher et al., 2008 4 372 4 (100) 143 (38) - 4/O:3 (142), O:9 (1) Gürtler et al., 2005
Great Britain 45 630 31 (69) 278 (44) - 2/O:5 (97), 2/O:9 (124), 4/O:3 (39) Martinez et al., 2010 - 2107 - - 85 (4) 2/O:9 (1), 3/O:5,27 (52), 3/O:9 (13),
4/O:3 (19) Milnes et al., 2008
- 2509 - - 246 (10) 3/O:5,27 (142), 3/O:9 (71), 4/O:3 (33)
McNally et al., 2004
Greece - 455 - 58 (13) - 4/O:3 Kechagia et al., 2007 Italy 22 428 22 (100) 137 (32) - 2/O:5 (1), 4/O:3 (136) Martinez et al., 2011
25 98 NS - 4 (4) 3/O:9 Bonardi et al., 2007
Country Number of farms
Number of pigs
Positive batches
(%)
Number of positive pigs
Bioserotypes Reference Positive tonsils
(%)
Positive faeces
(%) Japan 96 1200 32 (33) - 89 (7) 2/O:5,27 (1), 3/O:3 (43), 4/O:3 (45) Fukushima et al., 1989b Latvia 47 404 35 (74) 143 (35) - 4/O:3 Terentjeva and Berzins, 2010
5 109 3 (60) 70 (64) - 4/O:3 Martinez et al., 2009 Lithuania 11 110 6 (55) - 20 (18) 4/O:3 Novoslavskij et al., 2013 Norway 66 461 NS 67 (15) - 4/O:3 Nesbakken and Kapperud, 1985 Russiab 10 197 10 (100) 66 (34) - 4/O:3 Martinez et al., 2009 Spain 14 200 14 (100) 185 (93) - 4/O:3 Martinez et al., 2011
Switzerland 16 212 NS 72 (34) - 2/O:5,27 (6), 2:O:9 (1), 4/O:3 (69) Fredriksson-Ahomaa et al., 2007
aNS: not specified bRussia: Leningrad Region
GENERAL INTRODUCTION ________________________________________________________________________
________________________________________________________________________
35
5.3. Presence and control on pig farms
When the number of farms was taken into account in the study, there happened to be a
range from 33 to 100% positive farms (Table V). Sampling the same farms again overtime
showed that both enteropathogenic species can persist on farms (Skjerve et al., 1998;
Pilon et al., 2000; Niskanen et al., 2008; Poljak et al., 2010). In these positive farms, the
number of infected fattening pigs per farm varied (Fukushima et al., 1983; Letellier et al.,
1999; Gurtler et al., 2005; Laukkanen et al., 2009; Novoslavkij et al., 2013).
The spread within a pig farm can be caused by environmental factors or by pig-to-pig
transmission, but this has not been investigated thoroughly (Fukushima et al., 1983; Pilon
et al., 2000; Skjerve et al., 1998). Human pathogenic Y. enterocolitica has been isolated
from pig house structures such as floors, pen walls, hallways and stairs (Aldova et al.,
1980; Pilon et al., 2000; Bolton et al., 2013; Nathues et al., 2013). They were also found
on water-dependent materials like nipple drinkers, suckling devices and piping (Pilon et
al., 2000; Nathues et al., 2013). Al last, they were recovered on boots (Pilon et al., 2000;
Vilar et al., 2013). Yersinia pseudotuberculosis has only been detected from the pen floor
of fattening pigs (Niskanen et al., 2008). Neither of the pathogens was found in the
garbage or in the ventilation (Fukushima et al., 1983; Nathues et al., 2013).
The spread between pig farms can originate from incoming, infected piglets that will infect
the whole fattening pig unit (Virtanen et al., 2012). Similar genotypes were found
between farms that transported piglets between each other (Virtanen et al., 2014).The
environment of pig farms was studied for presence of enteropathogenic Yersinia spp.
They were found more often in the proximity of high infected farms (Fukushima et al.,
1983; Pilon et al., 2000).
Studies performed on a limited number of pig farms or pigs per farm sampled to search
for factors influencing this variation of the prevalence between farms or type of farms,
were already conducted (Table VI). Some factors concerning farm management are hard
to change and their attributed influence differs between studies. Only Christensen et al.
(1980), Skjerve et al. (1998) and Nesbakken et al. (2003) showed a higher prevalence of Y.
GENERAL INTRODUCTION ________________________________________________________________________
________________________________________________________________________
36
enterocolitica O:3 in fattening pig farms than in farrow-to-finish farms. Other studies did
not find this relation (Andersen et al., 1991; Laukkanen et al., 2010b; Wesley et al., 2008).
Also, the influence of the production type (conventional or organic) of pig farms on the
prevalence of both enteropathogenic Yersinia spp. has been studied many times, but
without a clear outcome. A farm was defined as organic according to the Commission
Regulation 889/2008 (EC 889/2008). The difference between conventional and organic
pig production concerning the prevalence of enteropathogenic Yersinia spp. has already
been studied in countries with a reliable number of organic pig farms. At pig level, Nowak
et al. (2006) found Y. enterocolitica in 36 out of 200 examined pigs of organic pig
production farms and in 60 out of 210 pigs from conventional housing systems. Virtanen
et al. (2011) suggested that the low prevalence of Y. enterocolitica in organic farms is
affected by generous use of bedding, limited use of antibiotics and lower animal density.
Pigs produced in organic farms have a lower daily weight gain (DWG) and are therefore
slaughtered at an older age, which implies less Y. enterocolitica in their tonsils and faeces
at time of slaughter, as mentioned above (Nielsen et al., 1996; Nesbakken et al., 2006).
Yersinia pseudotuberculosis is more common in organic production systems than in
conventional systems (Laukkanen et al., 2008; Ortiz-Martinez et al., 2010). Outdoor access
can provide different bioserotypes of enteropathogenic Yersinia spp. to pigs when contact
with wild animals occur (Niskanen et al., 2003). Martinez et al. (2010) found a variety of
different bioserotypes in English pigs, which could be due to a wider diversity of
enteropathogenic Yersinia spp. in English wild animals, subsequently transmitted to pigs.
A higher production capacity implies a higher prevalence of both Y. enterocolitica and Y.
pseudotuberculosis, due to underlying risk factors, for example larger group sizes and the
use of troughs for drinking (Laukkanen et al., 2008; Laukkanen et al., 2009; Laukkanen et
al., 2010b).
Measures influencing the prevalence of enteropathogenic Yersinia spp. on pig farms and
which are easy to apply do also exist. The factors underlying the measures influencing Y.
enterocolitica are first discussed, followed by those influencing Y. pseudotuberculosis, and
finally those influencing both species. Regarding Y. enterocolitica, the farm management
is an important factor. Purchasing piglets from more than one farm augments the
GENERAL INTRODUCTION ________________________________________________________________________
________________________________________________________________________
37
infection rate on farms. Fattening pig farms should therefore buy their piglets from Y.
enterocolitica-negative multiplying farms or from just one farm. This declines the risk of
introducing Y. enterocolitica to the farm (Virtanen et al., 2012; Virtanen et al., 2014). All-
in/all-out management seems to bring down the spread in pigs compared to continuous
production (Vilar et al., 2013). Secondly, housing also plays a role in the prevalence on pig
farms. Snout contact between pigs from adhering pens and the use of bedding material
increases the prevalence of Y. enterocolitica isolated from faeces or tonsils (Laukkanen et
al., 2009; Vilar et al., 2013; Virtanen et al., 2011). A third group of factors are feed related.
The use of commercial feed and industrial by-products is related to a higher occurrence
of Y. enterocolitica in pig herds (Nowak et al., 2006; Virtanen et al., 2011). In the U.S. the
use of meat or bone meal in slaughter pig diet is still allowed and is associated with a
higher prevalence (Wesley et al., 2008). The administration of a prebiotic element in the
diet of piglets helps dropping the occurrence of Y. enterocolitica (Virtanen et al., 2011).
The use of municipal water compared to collected water (rain, ground or well water) is
also a protective factor, probably the bacteriological level is controlled more frequently
(Virtanen et al., 2011; von Altrock et al., 2011; Vilar et al., 2013). Fourthly, there are
factors correlated with the health status of pig farms. Farms with a higher prevalence of
Y. enterocolitica were using antibiotics frequently due to recurring health problems, had
a lower DWG, applied vaccination against E. coli and had a higher percentage of deaths
due to scours (Wesley et al., 2008; Virtanen et al., 2011; von Altrock et al., 2011). These
high-prevalence farms were mostly categorized in the low Salmonella risk herd (Nathues
et al., 2013), while a lower prevalence was found in farms with a positive Salmonella
status (von Altrock et al., 2011). Wesley et al. (2008) did not find any relation with the
presence of Salmonella, roundworms, gastric ulcers, hemolytic bowel syndrome or ileitis.
At last, the presence of Y. enterocolictica is correlated with the access of pets and pest
animals to the stables (Wesley et al., 2008; Laukkanen et al., 2009; Virtanen et al., 2011).
These animals possibly spread and maintain infections of Y. enterocolitica on the farm.
Isolates collected on farm originating from pigs and pest animals possessed the same
genotypes (Aldova et al., 1980; Backhans et al., 2011). Farms dogs did not carry Y.
enterocolitica (Gürtler et al., 2005; Niskanen et al., 2008). Only one study examined
factors that were only correlated with the presence of Y. pseudotuberculosis (Laukkanen
GENERAL INTRODUCTION ________________________________________________________________________
________________________________________________________________________
38
et al., 2008). The only factor that is easy to apply was the contact with pest animals and
the outside environment. Risk factors mentioned in studies concerning both
enteropathogenic species are recurring health problems, a lower DWG and a large
number of pest animals in the stables (von Altrock et al., 2011; Novoslavkij et al., 2013).
These factors have already been mentioned in studies concerning only Y. enterocolitica.
Some factors were not significant according to previous studies, although they should
have an influence on the prevalence. An example of such a factor is the cleaning and
disinfection of the piggery that does not have an effect on the occurrence of both Y.
enterocolitica and Y. pseudotuberculosis on pig farms (Laukkanen et al., 2008; Virtanen et
al., 2011).
The presence of Y. enterocolitica and Y. pseudotuberculosis can be influenced actively by
applying bacteriocins and bacteriophages, vaccination or competitive exclusion. All of
these methods should be studied further. Bacteriocins isolated from Y. kristensenii have
already been tested on their influence on pathogenic Y. enterocolitica (Toora et al., 1994).
Also the bacteriocin enterocoliticin, isolated from Y. enterocolitica biotype 1A, was tested
on its suppression on the growth of pathogenic Y. enterocolitica (Strauch et al., 2001). This
bacteriocin has shown effect in vitro, unfortunately, it did not prevent colonization of the
intestinal tract by Y. enterocolitica (Damasko et al., 2005). The use of bacteriophages to
control infection with Y. enterocolitica in animals and humans has been studied (Skurnik
and Strauch, 2006). Yersinia pseudotuberculosis sometimes causes clinical disease in
animals. For this reason, vaccines against this pathogen have been developed for
administration in zoo and wildlife park animals, maras, deer and horses (Thornton and
Smith, 1996; Czernomysy-Furowicz et al., 2010; Quintard et al., 2010). Pseudovac®, a
killed whole cell vaccine, is the only registered vaccine in Europe (Y. pseudotuberculosis
serotypes O:1-O:6), and its manufacturer, the Department of Veterinary Pathology,
Utrecht University, The Netherlands, recommends to perform two subcutaneous
injections per year (Quintard et al., 2010). A second vaccine used for deer farms in New
Zealand is also a killed vaccine (Yersiniavax®, MSD Animal Health). However, vaccination
against the enteropathogenic Yersinia spp. has not yet been developed for pigs. The
GENERAL INTRODUCTION ________________________________________________________________________
________________________________________________________________________
39
studies around competitive exclusion linked with Yersinia are very limited. Hussein et al.
(2003) has found that Y. enterocolitica biotype 1A can bring down the adhesion of Y.
enterocolitica bioserotype 4/O:3 when the latter arrives later at the specific cells.
Unluckily, this happens only in vitro, not in vivo.
Table VI: Risk and protective factors for presence of enteropathogenic Yersinia spp. in pig herds.
Risk factor (RF) or protective factor (PF) Farms Pigs Type of samples
Country
Sfa Pb %c Spd P % Reference
Y. pseudotuberculosis
organic production (RF) high production capacity (RF) contact with pest animals and the outside environment (RF)
10-5 3-3 30-60 239-119 13-11 5-9
intestinal content
Finlande
Laukkanen et al., 2008 231-119 6-28 3-24 tonsil
Y. enterocolitica
own vehicle for transport of slaughter pigs to abattoirs (RF) separation between clean and unclean section in herds (RF) daily observations of a cat with kittens on the farm (RF) straw bedding for slaughter pigs (RF) farrow-to-finish production (PF) under-pressure ventilation (PF) manual feeding of slaughter pigs (PF)
179-86
95-74
53.1-86
1325 313-284
35-66 blood Norway f,g
Skjerve et al., 1998
conventional housing system (RF) sourcing pigs from different pig suppliers (RF) use of commercial feed (RF)
6-3 6-3 100 210-200
46-22 22-11 tonsils
Germanye Nowak et al., 2006
22-10 11-5 caecal content
14-4 7-2 caecal
lymph nodes
location in a central state (RF) vaccination for E. coli (RF) percentage of deaths due to scours (RF) presence of meat or bone meal in grower-finisher diet (RF)
100 124
32 62
32 50
1218 122 372
10 13.1
tonsils faecal samples
U.S. Wesley et al.,
2008
Risk factor (RF) or protective factor (PF) Farms Pigs Type of samples
Country
Sfa Pb %c Spd P % Reference
Y. enterocolitica
drinking from a nipple (RF) access of pest animals to pig house (PF) coarse feed or bedding for slaughter pigs (PF)
10-5 8-4 80-80
239-119 26-1 10-1 intestinal content Finlande
Laukkanen et al., 2009 231-119 106-18 46-15 tonsil
artificial light (h/day) (RF) daily/weekly use of antibiotics (RF) industrial by-products in feed (RF) tonsillar carriage (IC) (RF) feed from company B (IC) (RF) fasting pigs before transport to the slaughterhouse (IC) (RF) higher-level farm health classification (IC) (RF) snout contact (IC) (RF) use of tetracycline (IC) (RF) use of municipal water (PF) organic production (PF) buying feed from company A (PF) use of amoxicillin (IC) (PF)
85 87 94.2 519 318
15.4 intestinal
content (IC)
Finlandf Virtanen et al.,
2011
47.1 tonsils
pens without or with sparse amounts of bedding (RF) buying piglets from more than one farm (RF) using an all-in/all-out (PF) use of municipal water (PF)
26 - - 994 323 32.5 faecal samples Finlandh
Vilar et al., 2013
Risk factor (RF) or protective factor (PF) Farms Pigs Type of samples
Country
Sfa Pb %c Spd P % Reference
Y. pseudotuberculosis and Y. enterocolitica
low biosecurity level (RF) a large number of pest animals and pets (RF)
11 6-4 64-45 110h 20-11 h 18-10 h feacal samples Lithuaniai
Novoslavskij et al., 2013
more recurring health problems (RF) lower daily weight gain (RF) fully slatted floor (PF) use of municipal water (PF)
80 67 83.7 2400 1540 64.1 blood Germanyf
von Altrock et al., 2011
aSf: number of farms sampled bP: number of positive samples c%: percentage of positive samples dSp: number of pigs sample
e: conventional housing - alternative, organic housing f: only the number of farms and pigs that were used in the risk factor analysis are given
g: farrow-to-finish herds - slaughter pig production h: sows and boars were not included; multiplying farms not included i: Y.
enterocolitica 4/O:3 -Y. pseudotuberculosis 2/O:3
________________________________________________________________________
43
AIMS OF THE THESIS
AIMS OF THE THESIS ________________________________________________________________________
________________________________________________________________________
45
The consumption of raw or undercooked pork is the most important route of human
infection with enteropathogenic Y. enterocolitica. A possibility of pork getting
contaminated on the slaughter line is the (cross)contamination from tonsils and faeces of
infected pigs. The risk of (cross)contamination can be decreased by implementing better
hygienic measurements in the slaughterhouse and by reducing the number of infected
pigs arriving at the slaughterhouse. In Belgium, there is no information available about
the prevalence, the number of infected farms or the factors influencing this prevalence.
The general aim of this thesis is to gain insight into the variation of the within-batch
prevalence of enteropathogenic Yersinia spp. in pigs at slaughter age originating from
different farms and the factors influencing this prevalence.
Therefore, specific objectives of this thesis are:
- to investigate the microbiological and serological within-batch prevalence of
human pathogenic Y. enterocolitica and Y. pseudotuberculosis at the time of
slaughter and the variation of this prevalence (Chapter 1, 2 and 3)
- to evaluate the microbiological and serological prevalence data, in order to find a
comparison between these prevalences and to predict the infection status of pig
batches prior to slaughter (Chapter 4)
- to determine risk and protective factors influencing the infection at the moment
of slaughter based on microbiological and serological data (Chapter 5 and 6)
________________________________________________________________________
47
EXPERIMENTAL DESIGN
EXPERIMENTAL DESIGN ________________________________________________________________________
________________________________________________________________________
49
This research was based on two sampling periods.
1. First, a preliminary study to estimate the microbiological within-batch prevalence
of enteropathogenic Yersinia spp. in tonsils was performed. The number of pigs to
be sampled per batch was based on an expected batch prevalence of 50%, a
confidence level of 95% and an accepted error of 20% (Chapter 1). The
microbiological within-batch prevalence showed a large variation, without
clustering around certain percentages.
2. This was the basis of the second and more elaborate sampling period. During these
studies, the within-batch prevalence examining both tonsils (Chapter 2) and pieces
of diaphragm (Chapter 3) was investigated. The accepted error was adapted to
10%, resulting in more pigs per batch to be sampled. Yersinia enterocolitica and Y.
pseudotuberculosis could be analyzed separately in the study of the
microbiological prevalence, while there is no distinction between the two species
in the serological study. The microbiological study determines the infection status
of the batch at moment of slaughter, while serology at moment of slaughter
presents an indication of previous infections. The comparison of both matrixes
(tonsil and meat juice) originating from the same pig could be useful to identify
microbiologically positive batches based on serology (Chapter 4). Finally, the
results of the microbiologically and serologically based within-batch prevalence
were used in two separate risk factor analyses. The farms delivering the batches
sampled in the slaughterhouse were visited prior to sampling and a questionnaire
was filled in. The risk factor analysis based on the microbiological within-batch
prevalence of Y. enterocolitica (Chapter 5) identifies the factors influencing the
presence of this single pathogen in the tonsils at moment of slaughter. The risk
factor analysis based on the serological within-batch prevalence of both Y.
enterocolitica and Y. pseudotuberculosis (Chapter 6) points out the factors
influencing the risk of infection on-farm.
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51
CHAPTER 1
ESTIMATION OF THE WITHIN-BATCH PREVALENCE
AND QUANTIFICATION OF HUMAN PATHOGENIC
YERSINIA ENTEROCOLITICA IN PIGS AT SLAUGHTER
Modified from: Vanantwerpen, G., Houf, K., Van Damme, I., Berkvens, D., De Zutter, L.,
2013. Estimation of the within-batch prevalence and quantification of human pathogenic
Yersinia enterocolitica in pigs at slaughter. Food Control 34, 9-12.
CHAPTER 1 ________________________________________________________________________
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52
1. Abstract
Yersiniosis is the third most common of bacterial zoonosis in the EU. The main source for
human infection is pork contaminated with human pathogenic Yersinia enterocolitica, for
which pigs are the primary reservoir. The aim of this study was to acquire data about the
distribution of the prevalence of human pathogenic Y. enterocolitica in different batches
of slaughter pigs. Between August and October 2011, in five Belgian slaughterhouses
tonsils of 1397 fattening pigs, originating from 66 batches, were collected. Samples were
plated onto cefsulodin-irgasan-novobiocin agar plates and suspect Yersinia colonies were
enumerated. Y. enterocolitica were found in 375 pig tonsils (26.8%), originating from 46
batches. The within-batch prevalence showed a large variation between the different
batches and ranged from 0 to 83.3%. In 20 batches (30.3%), no positive tonsils were
detected. The average number of Y. enterocolitica was 4.04 ± 0.97 log10 CFU g-1 tonsillar
tissue and the mean Yersinia count per batch varied between 3.08 and 5.89 log10 CFU g-1.
In conclusion, human pathogenic Y. enterocolitica is widespread among Belgian pig farms,
but there is a large variation in the within-batch prevalence among farms.
CHAPTER 1 ________________________________________________________________________
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53
2. Introduction
Yersiniosis is the third most common bacterial zoonosis in the EU, after
campylobacteriosis and salmonellosis (EFSA and ECDC, 2013). Based on qualitative risk
analysis of foodborne hazards in pork, Yersinia enterocolitica is considered as a hazard of
medium relevance for human health in the EU (Biohaz, 2011). The average EU incidence
of yersiniosis is 1.63/100 000 inhabitants. In 2011, Y. enterocolitica caused almost 91% of
the 7017 reported cases of human yersiniosis in Europe (EFSA and ECDC, 2013). It mainly
affects young children of 1-4 years old. Typical symptoms are fever, diarrhea and
abdominal pain (Bottone, 1997).
The five human pathogenic Y. enterocolitica biotypes (1B, 2-5) possess chromosomal and
plasmid-associated virulence factors, including respectively the ail and virF genes. The
species can also be characterized by serotyping, however without virulence
determination (Bottone, 1999). The most often detected bioserotype in humans is 4/O:3
(83%), followed by 2/O:9 (15%) (EFSA and ECDC, 2013).
The consumption of pork is the main source for human infection and healthy pigs are
known to be the primary reservoir of Y. enterocolitica (Tauxe et al., 1987; Bottone, 1999;
Fosse et al., 2009; Huovinen et al., 2010; EFSA and ECDC, 2013). Other production animals
like ruminants can also be infected, but they seldom carry bioserotype 4/O:3 (Nikolova et
al., 2001; Carter and Wise, 2003). Pigs are potential asymptomatic carriers of human
pathogenic Y. enterocolitica in the tonsils and the intestines, which can lead to
contamination of the carcass during slaughter (Nesbakken, 1985; Bottone, 1999;
Nesbakken 2000; Fredriksson-Ahomaa et al., 2001a; Simonova et al., 2008). Because both
ante- and post-mortem meat inspection of pigs currently do not target this
contamination, it is suggested that new appropriate procedures should be developed. The
occurrence and the level of contamination of Y. enterocolitica on pig carcasses are highly
variable depending on the origin and the occurrence in pigs prior to slaughter (Biohaz,
2011). A reduction of the prevalence of human pathogenic Y. enterocolitica on the pig
farms could decrease the (cross-) contamination of the carcasses at the slaughterhouse
CHAPTER 1 ________________________________________________________________________
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54
and eventually also the pork meat (Laukkanen et al., 2009). This is also the concept of the
future European meat inspection, so the Y. enterocolitica status should be known before
the pigs arrive at the slaughterhouse (Biohaz, 2011). Serological methods can indicate if
pigs have ever been exposed to Y. enterocolitica but not a current infection, so the
prevalence at slaughter should be established by microbiological examination of tonsils
or faeces. The tonsils at time of slaughter are possibly a more significant source of human
pathogenic Y. enterocolitica than faeces due to a higher number of pigs infected in the
tonsils and the higher counts of Yersinia spp. in the tonsils than in the faeces (Nesbakken
et al., 2003; Nesbakken et al., 2006; Van Damme, 2013).
The overall-prevalence of Y. enterocolitica in tonsils has already been determined
extensively in different countries (Fredriksson-Ahomaa et al., 2000a; Bonardi et al., 2003;
Fredriksson-Ahomaa et al., 2007; de Boer et al., 2008; Simonova et al., 2008; Martinez et
al., 2009; Poljak et al., 2010). However, only few studies have reported the within-herd
prevalence so far (Skjerve et al., 1998; Nowak et al., 2006; Virtanen et al., 2011). For the
risk categorization of slaughter pig batches, the within-herd prevalence should be
monitored and new data about the current prevalence at pig farms should be provided
(Biohaz, 2011). The aim of this study was to obtain data about the distribution of the
prevalence of Y. enterocolitica in Belgian slaughter pig batches.
3. Materials and methods
3.1. Sampling
Sixty-six batches, each originating from different pig herds, were examined in five Belgian
slaughterhouses between August and October 2011. All batches originated from fattening
and farrow-to-finish herds. Fattening pigs were slaughtered at an age of 6 to 6.5 months
and had an average slaughter weight of 120 kg. The five selected slaughterhouses
slaughtered 450 to 600 pigs per hour. The time of transport varied from 30 minutes to 2
hours and all pigs were slaughtered between 30 and 90 minutes after arrival.
CHAPTER 1 ________________________________________________________________________
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55
Due to the current lack of information about the Y. enterocolitica prevalence in pig
batches, the sample size per batch was calculated based on an expected batch prevalence
of 50%, a confidence level of 95% and an accepted error of 20%.
The tonsils (tonsilla veli palatini) were aseptically removed from the head immediately
after the removal of the pluck and placed in a sterile stomacher bag. All samples were
transported under cooled conditions to the laboratory and examined within 3 h after
arrival.
3.2. Detection and enumeration of human pathogenic Y. enterocolitica
The tonsils (1.0-10.0g) were divided into small pieces, diluted in 0.1% peptone water
(1/10, w/v) and homogenized in a stomacher blender (Colworth Stomacher 400, Seward,
London, U.K.) for 2 min. One hundred microliter from each homogenate was plated onto
a cefsulodin-irgasan-novobiocin (CIN) agar plate (Yersinia Selective Agar Base and Yersinia
Selective Supplement, Oxoid, Basingstoke, UK) with a spiral plate machine (Eddie Jet, IUL
Instruments, Barcelona, Spain), allowing the detection as well as the enumeration of Y.
enterocolitica. After incubation at 30°C for 24 h, all CIN plates were examined using a
stereo microscope with Henry illumination (Olympus, Aartselaar, Belgium) and the
number of suspect Yersinia colonies (typically bull’s eye colonies with a red centre) was
counted. From each plate, one suspect colony was streaked onto a Plate Count Agar (PCA)
plate (Bio-Rad, Marnes-La-Coquette, France) and incubated at 30°C for 24 h. Then,
cultures were transferred into Urease Broth (Ur), Kligler Iron Agar (KIA) (Oxoid) and
Tryptone Soy Broth (TSB) (Bio-Rad) and incubated at 30°C for 24 h. When fermentation of
glucose, no fermentation of lactose and no development of gas or H2S on KIA and a
degradation of urea was observed, the isolates were considered as presumptive Y.
enterocolitica and were subsequently confirmed using a Polymerase Chain Reaction (PCR)
to detect the ail gene (Harnett et al., 1996). One hundred microliter from each TSB culture
was centrifuged at 12 000 rpm for 2 min. The supernatant was removed, 50 μl PrepMan®
Ultra (Applied Biosystems, Foster City, U.S.) was used to suspend the pellet and the tubes
were put in a heat-block (Grant, Cambridge, U.K.) for 10 min at 100°C. After 2 min of
CHAPTER 1 ________________________________________________________________________
________________________________________________________________________
56
cooling down, the tubes were again centrifuged at 12 000 rpm for 2 min. Thirty microliter
of the supernatant was stored at -20°C as DNA template in the PCR assay. One microliter
of the template was added to 24 μl of the PCR mix (Promega, Leiden, The Netherlands),
which contained the ail primer set (Invitrogen, Paisley, U.K.).
3.3. Statistical analysis
Results were recorded in an Excel spreadsheet and the quantitative data were log
transformed. Only the samples with countable numbers were taken into account to
calculate the mean count for each batch. To identify the limit of detection of infection in
batches without any positive tonsil sample, Win Episcope was used (Thrusfield et al.,
2001).
4. Results
Samples were collected from 1397 fattening pigs originating from 66 batches. The size of
the batches varied from 34 to 930 animals, with a mean batch size of 228. The number of
pigs to be sampled varied from 17 to 24, with an average of 21 pigs per batch.
In total, ail gene positive Y. enterocolitica was recovered from 375 pig tonsils (26.8%; 95%
confidence interval (CI): 24.5-29.1%). The within-batch prevalence showed a large
variation between the different batches and ranged from 0 to 83.3% (Fig. II). The data
presented a bimodal distribution, with modes at the classes 0% (20/66) and 35-39.9%
(9/66). In 69.7% of all slaughter pig batches at least the tonsils of one pig was found
positive. In 20 batches (30.3%), no Y. enterocolitica was isolated from the tonsils. Based
on an average of 21 samples per batch, the upper 95% CI limit for the prevalence of
negative batches was 12.7%.
CHAPTER 1 ________________________________________________________________________
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57
The average number of Y. enterocolitica in tonsil tissue was 4.04 log10 CFU g-1 with a
standard deviation of 0.97 log10 CFU g-1 and a maximum of 5.99 log10 CFU g-1 (Fig. III). In
14 samples, the number of colonies could not be counted due to overgrowth of
accompanying flora. Seventy of the 375 Yersinia positive tonsils (18.7%) contained
between 5.00 and 5.49 log10 CFU g-1. Eight tonsils (0.02%) were contaminated over 5.49
log10 CFU g-1 tonsillar tissue.
0
2
4
6
8
10
12
14
16
18
20
num
ber
of
bat
ches
prevalence (%)
Figure II. Frequency distribution of the presence of Y. enterocolitica in batches (n=66) of
slaughter pigs.
CHAPTER 1 ________________________________________________________________________
________________________________________________________________________
58
The mean Yersinia count per batch varied between 3.08 and 5.89 log10 CFU g-1 tonsillar
tissue with a standard deviation of 0.54 log10 CFU g-1 (Fig. IV). Out of the 46 infected
batches, most (n=18) had a contamination level between 4.00-4.49 log10 CFU g-1.
0
10
20
30
40
50
60
70
80num
ber
of
Y. en
tero
coli
tica
posi
tive
tonsi
ls
Yersinia count (Log10 CFU g-1 tonsillar tissue)
0
2
4
6
8
10
12
14
16
18
20
3.00-3.49 3.50-3.99 4.00-4.49 4.50-4.99 5.00-5.49 5.50-5.99
num
ber
of
Y. en
tero
coli
tica
posi
tive
bat
ches
mean Yersinia count (Log10 CFU g-1 tonsillar tissue)
Figure III. Frequency distribution of Y. enterocolitica counts in tonsillar tissue.
Figure IV. Frequency distribution of the mean Y. enterocolitica counts in
tonsillar tissue per pig batch.
CHAPTER 1 ________________________________________________________________________
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59
5. Discussion
In consequence of the concept of the future European meat inspection, determining the
microbiological prevalence in batches could allow division of farms into different risk
groups (Biohaz, 2011).
Nonetheless, the information about the distribution of the prevalence of human
pathogenic Y. enterocolitica within pig batches at slaughter is limited. Therefore the
number of samples to be taken per herd was determined at the beginning of this study,
based on an expected batch prevalence of 50%. In 20 pig herds (30.3%), no infected pigs
were found. Nevertheless, batches with a prevalence below 12.7% might not be detected
due to the sample size used. Moreover, only tonsil samples were examined in the present
study, as tonsils are the most reliable samples to detect Y. enterocolitica in pigs
(Nesbakken et al., 2003; Gürtler et al., 2005). Therefore, information about other tissues
or organs could have resulted in more positive batches. Hence, we have no information
whether the negative batches in the present study are indeed free of Y. enterocolitica or
have a prevalence below the detection limit.
In the present study, 69.7% of the batches was positive for human pathogenic Y.
enterocolitica, which is within the range of other studies. The number of positive batches
varies between 15 and 94% among different studies (Skjerve et al., 1998; Martinez et al.,
2010; Poljak et al., 2010; Terentjeva and Berzins, 2010; Virtanen et al., 2011). Several
factors may attribute to this variation, such as the number of samples per herd, the
sample matrix and the test method. Skjerve et al. (1998) found pigs with positive serum
samples in 63% of the herds in Norway analyzing 5 to 43 pigs per herd. Virtanen et al.
(2011) isolated Y. enterocolitica from 94% of Finnish farms testing the tonsils and faeces
of 2 to 86 pigs per herd.
The prevalence within batches in the current study varied from 0 to 83.0%. Similarly,
Gürtler et al. (2005) examined pig tonsils of six batches at slaughter, which resulted in a
within-batch prevalence between 8 and 69%. Based on serology, von Altrock et al. (2011)
CHAPTER 1 ________________________________________________________________________
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60
investigated 80 farms (30 samples per herd) which resulted in a within-batch prevalence
of 0 to 100%. In contrast, Nowak et al. (2006), found only a small number of pigs per farm
to be infected with Y. enterocolitica in the tonsils. Except for the study of Nowak et al.
(2006), the within-batch prevalence seems to vary greatly among different farms.
Destructive tissue samples were used in this study as swabbing the tonsil has already been
shown to be insufficient for the detection of Y. enterocolitica in pig tonsils (Nesbakken et
al., 1985; Van Damme et al., 2012). Both studies yielded more positive tonsillar tissue
samples compared to swab samples, although each study applied different isolation
methods. Moreover, direct plating was used as the only isolation method as it has already
been shown to be an adequate method for isolation of Y. enterocolitica from pig tonsils
and additionally quantitative data can be obtained (Van Damme et al, 2010). Moreover,
because the main goal of the present study was to gain insight in the distribution of the
prevalence in pigs at slaughter, positive samples due to cross-contamination occurring at
the slaughterhouse has to be excluded as much as possible.
To our knowledge, for the first time, quantitative data per herd was obtained. Highly
contaminated pigs are more likely to cause cross-contamination to the carcass. There is a
low within-batch prevalence observed when there is a high mean count. This might be
explained by the moment of infection (Nesbakken et al., 2006). A recent infection of pigs
might cause colonization of a limited number of pigs within a group, leading to high
Yersinia count in the tonsils. This hypothesis has to be confirmed by serology, since recent
infected animals would lack antibodies.
In conclusion, there is a wide distribution of the prevalence of human pathogenic Y.
enterocolitica in batches of pigs at slaughter. Therefore it may be interesting to include in
risk factor studies beside the Y. enterocolitica status of the batches of pigs also the
prevalence in the batches. In such studies sufficient samples have to be collected in order
to estimate the prevalence more accurately so that potential risk factors can be better
linked to the observed prevalences in the studied batches.
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61
CHAPTER 2
WITHIN-BATCH PREVALENCE AND
QUANTIFICATION OF HUMAN PATHOGENIC
YERSINIA ENTEROCOLITICA AND Y. PSEUDOTUBERCULOSIS IN TONSILS OF PIGS AT
SLAUGHTER
Modified from: Vanantwerpen, G., Van Damme, I., De Zutter, L, Houf, K.., 2014. Within-
batch Prevalence and quantification of Human Pathogenic Yersinia enterocolitica and Y.
pseudotuberculosis in Tonsils of Pigs at Slaughter. Veterinary Microbiology 169, 223-227.
CHAPTER 2 ________________________________________________________________________
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62
1. Abstract
Yersiniosis is a common bacterial zoonosis in Europe and healthy pigs are known to be the
primary reservoir of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis.
However, little information is available about the prevalence of these pathogens within
pig batches at time of slaughter. The tonsils of 7047 fattening pigs, belonging to 100 farms,
were aseptically collected immediately after evisceration in two Belgian slaughterhouses.
The batch size varied between 70 and 930 pigs. On average, 70 pigs were sampled per
batch. The tonsils were examined by direct plating on cefsulodin-irgasan-novobiocin (CIN)
agar plates and the number of suspect Yersinia colonies was counted. Pathogenic Y.
enterocolitica serotype O:3 were found in tonsils of 2009 pigs (28.5%), originating from 85
farms. The within-batch prevalence in positive farms ranged from 5.1 to 64.4%. The
number of Y. enterocolitica in positive pigs varied between 2.01 and 5.98 log10 CFU g-1
tonsil, with an average of 4.00 log10 CFU g-1 tonsil. Y. pseudotuberculosis was found in
seven farms, for which the within-batch prevalence varied from 2 to 10%. In five of these
farms, both Y. enterocolitica and Y. pseudotuberculosis were simultaneously present.
Human pathogenic Yersinia spp. are widespread in slaughter pig batches in Belgium as
87% of the tested batches were infected with these pathogens at time of slaughter. The
large variation of the prevalence between batches may lead to different levels of
contamination of carcasses and risks for public health.
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63
2. Introduction
Yersiniosis is a significant foodborne disease in Europe, primarily caused by the species
Yersinia enterocolitica (98%) and to a lesser account by Y. pseudotuberculosis. The
average EU incidence of yersiniosis is 1.63 per 100,000 inhabitants (EFSA and ECDC, 2013).
Clinical manifestation is symptomized by diarrhea, abdominal pain and fever and occurs
mostly in young children (Bottone, 1999). In children (> 4 years) and adults, pain in the
lower right abdomen and fever may be the predominant symptoms and therefore it is
often confused with appendicitis (Bottone, 1999).
The pathogenicity of Yersinia spp. is determined by the presence of virulence genes
encoded on the chromosome and on the pYV. The sequence of the chromosomally
encoded virulence genes ail, yst and inv are species-dependent and sometimes even vary
within one species. Different PCR based assays have been developed for the identification
of enteropathogenic Yersinia spp. with several PCR assays targeting the pYV-encoded
genes, such as yadA and virF (Miller et al., 1989; Cornelis, 1998; Bottone, 1999; Revell and
Miller, 2001).
The species Y. enterocolitica is divided into six biotypes (1A, 1B, 2-5), of which only biotype
1A does not carry pYV and is therefore considered apathogenic. Further differentiation
into serotypes is not biotype-dependent, though some combinations, such as
bioserotypes 4/O:3 and 2/O:9, are more frequently distributed in Europe and cause most
human infections (EFSA and ECDC, 2013).
Although many animal species may carry pathogenic yersiniae, pigs are known as the main
reservoir of human pathogenic Y. enterocolitica, especially of bioserotype 4/O:3 (EFSA and
ECDC, 2013). Yersiniae are predominantly present in pigs’ tonsils and are shed in the
faeces (Fredriksson-Ahomaa et al., 2001a). During slaughter, carcasses can be faecally
contaminated or contaminated by infected tissues of its own or its near neighbors. The
risk of (cross-)contamination in the slaughterhouse can be reduced by a decrease of the
within-batch prevalence (Laukkanen et al., 2009).
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64
In a qualitative risk analysis of foodborne hazards in pork performed by the European
Food Safety Authority (EFSA), Y. enterocolitica ranked the second most important pork-
related bacterial pathogen (Biohaz, 2011). EFSA proposed to categorize slaughter pig
batches in risk classes, in order to facilitate appropriate actions taken at slaughterhouse-
level. Therefore, information regarding the within-herd prevalence is required (Biohaz,
2011).
Few studies have reported the within-herd prevalence so far. Skjerve et al. (1998) and von
Altrock et al. (2011) determined this within-batch prevalence serologically. The other
researchers used a microbiological pathway, using different sample matrices (Nowak et
al., 2006; Laukkanen et al., 2009, Fondrevez et al., 2010; Virtanen et al., 2011; Novoslavskij
et al., 2012; Chapter 1). As tonsils remain infected for a longer period than faeces, the
within-batch prevalence of pigs at slaughter is preferably assessed by microbiological
examination of tonsils rather than faeces (Nesbakken et al., 2006).
As little information is available about the prevalence of human pathogenic Y.
enterocolitica and Y. pseudotuberculosis within pig batches, the aim of this study was to
determine the within-batch prevalence of these pathogens in the tonsils of pigs at
slaughter.
3. Materials and methods
3.1. Sampling
From January until December 2012, in two Belgian slaughterhouses (A and B), tonsils from
7047 fattening pigs, representing 100 pig batches, were aseptically removed from the
head immediately after evisceration. All batches originated from 100 different
conventional farrow-to-finish and fattening pig farms. The pigs were slaughtered at an
age of 6 to 6.5 months with a live body weight of about 120 kg. The number of pigs to be
sampled per batch was calculated based on an expected batch prevalence of 50%, with a
CHAPTER 2 ________________________________________________________________________
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65
confidence level of 95% and an accepted error of 10%. The tonsils were transported under
cooled conditions to the laboratory and analyzed within the same day.
3.2. Detection and enumeration of human pathogenic Y. enterocolitica and Y.
pseudotuberculosis
The detection and enumeration are described in Chapter 1.
3.3. Confirmation by PCR
DNA was extracted as described by Van Damme et al. (2013). A first PCR targeting the
virulence genes ail, yst and virF was performed (Harnett et al., 1996). A second multiplex
PCR was processed to determine the serotype with rfbC and per primer sets (Weynants et
al., 1996; Jacobsen et al., 2005). When no fragments or only the fragment generated for
the virF was present, a third PCR assay was performed targeting the inv gene to identify
Y. pseudotuberculosis (Nakajima et al., 1992).
3.4. Statistical analysis
Results were registered in an Excel spreadsheet and the quantitative data were log
transformed. The detection limit of the direct plating method was 2 log10 CFU g-1 tonsil.
To calculate the mean count for each batch, only the samples with countable numbers
were taken into account. Win Episcope was used (Thrusfield et al., 2011) to identify the
limit of detection of infection in batches without any positive tonsil sample. A batch was
considered positive when at least one pig within the batch carried Y. enterocolitica or Y.
pseudotuberculosis in the tonsils. The overall prevalence and the 95% confidence interval
(CI) for Y. enterocolitica and Y. pseudotuberculosis was calculated using Stata/MP 12.1
(StataCorp, 2011), declaring batches as the primary sampling units (clusters).
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66
4. Results
Pigs from 100 farms were sampled in two Belgian slaughterhouses. The batch size ranged
from 70 to 930, with a mean of 314 ± 169 pigs per batch. The calculated sample size varied
from 41 to 88 pigs per batch (mean: 70 ± 10). In total, tonsils from 7047 pigs were
collected.
In 2009 tonsils (28.5%; 95% CI: 24.9-32.2%), ail- and yst-positive Y. enterocolitica serotype
O:3 was present (Table VII). In 85 of the 100 batches, at least one pig carried Y.
enterocolitica in the tonsils. The mean within-batch prevalence of positive batches was
33.5% ±17.6% and ranged from 5.1 to 64.4% (Fig. V). The data presented a bimodal
distribution, with modes at classes 0% (15/100) and 25-35% (22/100). The smallest and
largest negative batch (n=15) included 170 and 540 pigs respectively, which represents a
variation of the upper 95% CI limit for the prevalence in negative batches between 4.12
and 3.33%.
Table VII. Presence and quantification of human pathogenic Y. enterocolitica and Y. pseudotuberculosis in pigs (n=7047) and batches (n=100).
a: only counting for positive batches
Table VIII. Comparison between the two slaughterhouses.
Number of
positive pigs (%)
Quantification in positive tonsils (log10
CFU g-1 tonsillar tissue)
Number of positive
batches (%)
Within-batch prevalence (%)a
Quantification in positive batches (log10 CFU g-1
tonsillar tissue)
Mean Range Mean Range Mean Range
Y. enterocolitica Y. pseudotuberculosis
Human pathogenic Yersinia spp.
2009 (28.5) 23 (0.3)
2031 (28.8)
4.00 ±0.96 3.60 ±0.94 3.97 ±0.96
2.01-5.98 2.01- 5.49 2.01-5.98
85 (85.0) 7 (7.0)
87 (87.0)
33.5 ±17.6 5.3 ±25.3
33.2 ±17.2
5.1-64.4 2-10
4.1-64.6
3.96 ±0.29 3.55 ±0.61 3.92 ±0.29
2.91- 4.67 2.48-4.34 2.91-4.67
human pathogenic Y. enterocolitica Y. pseudotuberculosis
Sa Bb Pc Mean batch size(95% CI)
Proportion of positive pigs (95%
CI)
Number of positive batches
Mean within-batch
prevalenced (95% CI)
Proportion of positive pigs
(95% CI)
Number of positive batches
Mean within-batch
prevalenced
(95% CI)
A 50 3488 290 (251; 328) 29.0 (23.6; 34.5) 41 35.9 (5.4;66.4) 0.4 (0-0.9) 5 4.8 (-1.5;11.2) B 50 3559 337 (283; 392) 28.0 (23.2; 32.8) 44 31.3 (3.1;59.5) 0.1 (0-0.5) 2 6.4 (-3.2;15.9)
a: slaughterhouse b: number of batches c: number of pigs d: based on positive batches only
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68
The number of Y. enterocolitica in positive tonsils and positive batches is shown in Table
VII. In 87 samples, the number of colonies was not countable due to the overgrowth by
accompanying flora. The number of Y. enterocolitica in positive tonsils varied between
2.01 and 5.98 log10 CFU g-1 tissue, with a mean of 4.00 ± 0.96 log10 CFU g-1 tonsil. The
frequency distribution of Y. enterocolitica counts in the 2009 contaminated tonsils is
shown in Figure VI. At batch-level, the mean number ranged from 2.91 to 4.67 with an
overall mean of 3.96 ± 0.96 log10 CFU g-1 tonsil. The frequency distribution of Y.
enterocolitica mean numbers in positive batches is shown in Figure VII.
Only seven pig batches were positive for Y. pseudotuberculosis and showed a within-batch
prevalence ranging from 2 to 10% (Table VII). In 23 tonsils (0.33%; 95% CI: 0.05-0.60%),
inv-positive Y. pseudotuberculosis was present. From five batches, both Y. enterocolitica
and Y. pseudotuberculosis were isolated, though both pathogens were only
simultaneously present in one pig. In the 23 tonsils infected with Y. pseudotuberculosis,
the contamination level varied between 2.01 and 5.49 log10 CFU g-1 tonsillar tissue with an
0
2
4
6
8
10
12
14
16
num
ber
of
Y. en
tero
coli
tica
posi
tive
bat
ches
within-batch prevalence of Y. enterocolitica (%)
Figure V. Frequency distribution of the within-batch prevalence of Y.
enterocolitica in tonsils of slaughter pigs.
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69
average of 3.60 ± 0.94 log10 CFU g-1 tonsil. At batch level, the mean count of the Y.
pseudotuberculosis positive batches ranged from 2.48 to 4.34 log10 CFU g-1 tonsillar tissue,
with an average of 3.55 ± 0.61 log10 CFU g-1 tonsil.
Figure VI. Frequency distribution of Y. enterocolitica counts in tonsillar tissue.
0
50
100
150
200
250
300
350
400
450
num
ber
of
Y. en
tero
coli
tica
posi
tive
tonsi
ls
Yersinia count (log10 CFU g-1)
0
5
10
15
20
25
30
35
num
ber
of
Y. en
tero
coli
tica
posi
tive
bat
ches
mean Yersinia count (log10 CFU g-1)
Figure VII. Frequency distribution of the mean Y. enterocolitica counts in
tonsillar tissue per pig batch.
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70
Comparing the two slaughterhouses, the overall proportion of positive pigs was very
similar in both slaughterhouses (Table VIII). The mean batch size and the number of Y.
enterocolitica positive batches were slightly higher in slaughterhouse B than in
slaughterhouse A. The mean within-batch prevalence of Y. enterocolitica positive batches
was higher in slaughterhouse A, though no significant differences were observed.
In total, 3930 Y. enterocolitica O:3 isolates were collected, of which 260 (6.6%) showed no
degradation of urea. All of these urea-negative isolates harbored ail, yst, rfbC and most of
them (188/260) also virF. Only urea-negative isolates were present in three batches, 14
batches yielded both urea-positive and negative isolates whereas all isolates from 68
batches were able to degrade urea. Fifteen pig tonsils harbored urea-negative as well as
urea-positive isolates. One out of 46 Y. pseudotuberculosis isolates did not show
degradation of urea.
The presence of the pYV was verified by PCR targeting the virF-gene. Nine hundred and
three Y. enterocolitica isolates (23%) were virF-negative (Table IX). The prevalence of virF-
negative isolates per batch ranged from 0 to 60%. In seven batches all isolates were virF-
positive. Both types of isolates could be present in the same pig tonsil. Only 3 pig tonsils
harbored Y. pseudotuberculosis without virF.
Table IX. The presence of virF in Y. enterocolitica and Y. pseudotuberculosis at isolate-, pig-
and batch-level.
Y. enterocolitica Y. pseudotuberculosis
Isolate
(n=3930) (%)
Pig
(n=7047) (%)
Batch
(n=100)
(%)
Isolate
(n=45) (%)
Pig
(n=7047)
(%)
Batch
(n=100) (%)
virF - 903 (23.0) 572 (8.1) 78 (78.0) 5 (11.1) 3 (0.04) 3 (3.0)
virF + 3027 (77.0) 1688 (24.0) 85 (85.0) 40 (88.9) 21 (0.3) 6 (6.0)
Total 3930 (100) 2009 (28.5) 85 (85.0) 45 (100) 23 (0.3) 7 (7.0)
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71
5. Discussion
The present study demonstrates that fattening pig batches were frequently infected with
human pathogenic Y. enterocolitica O:3. However within a positive batch the number of
colonized pigs varied widely. Due to the possible cross-contamination in the
slaughterhouse, presence of a high proportion of infected pig batches at slaughter
represents a potential risk for public health (Nesbakken et al., 2003). The high prevalence
of enteropathogenic Y. enterocolitica at farm-level can result in a high contamination rate
of the carcass (Laukkanen et al., 2009).
The batch-level prevalence of batches positive for Y. enterocolitica ranged in the present
study from 5.1 to 64.4%, which is very similar to the result of Gurtler et al. (2005) (5.6 to
68.8%). This is in contrast to the analysis of Poljak et al. (2010) and Terentjeva and Bezins
(2010), where the within-batch prevalence ranged from 7 to 100% and from 0 to 100%
respectively. Several factors may attribute to this variation, such as the sample size (410-
2400), sample matrix (tonsils, faeces or blood) and the test method (PCR, cold enrichment,
ELISA). The variation in the within-batch prevalence in a study can be explained by the
variability in the farm management and within-farm factors, like the use of fully slatted
floors and municipal water (Nowak et al., 2006, Virtanen et al., 2011, von Altrock et al.,
2011).
In the present study, 15% of the batches were Y. enterocolitica negative which suggests a
widespread infection of pig batches at slaughter with Y. enterocolitica. This result is
comparable with the study of Martinez et al. (2011) which was performed in the same
region and where 2 of the 10 farms (20%) were Yersinia-free. Other studies reported an
amount of negative batches varying from 5.8% to 46.7% (Bhaduri et al., 2005; Martinez et
al., 2009; Terentjeva and Bezins, 2010; Virtanen et al., 2011; Novoslavskij et al., 2013).
In the current study the Belgian overall-prevalence of Y. enterocolitica was 28.5%. This
study was set up to determine the prevalence at batch-level. In Chapter 1, the pig-level
prevalence was 26.8%. The result is comparable to other studies reported in the same
CHAPTER 2 ________________________________________________________________________
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72
country. Van Damme et al, (2010) and Martinez et al. (2011) estimated this prevalence at
37.4% and 44% respectively. This variation could be due to a different study design
(determining within-batch prevalence by stratified random sampling versus overall-
prevalence by random sampling) and the isolation method (direct plating, selective and
cold enrichment: via enrichment procedures it is possible to detect low concentrations)
used. Between pig-producing countries a large variation is noticed when considering the
results of prevalence studies in Greece (13%), Lithuania (25%), Italy (32%), Switzerland
(34%), Latvia (35-64%), Finland (49.9%), Estonia (89%), Spain (93%), Canada (5.1-35.1%)
and the U.S. (13.1%) (Gürtler et al., 2005; Fredriksson-Ahomaa et al., 2007; Kechagia et
al., 2007; Martinez et al., 2009; Poljak et al., 2010; Terentjeva and Bezins, 2010; Martinez
et al., 2011; Virtanen et al., 2011; Novoslavskij et al., 2012).
Little information is available about the within-batch prevalence of Y. pseudotuberculosis.
In the current study, it ranged from 2 to 10%. This contrasts with the findings of Terentjeva
and Bezins (2010), were the prevalence peaked at 40% in one batch. Laukkanen et al.
(2008) found a within-farm prevalence up to 75%, and a higher prevalence in pig farms
with an organic compared to a conventional management. These different management
systems may have a different input of Yersinia spp. because organic systems are more
influenced by environmental conditions (climate, wild animals) than conventional
systems. In the present study, only conventional farms were included as organic farms are
very rare in Belgium. Due to this lower within-batch prevalence, a large number of
samples need to be taken to detect batches infected with Y. pseudotuberculosis than with
Y. enterocolitica. The Y. pseudotuberculosis negative batches were more common in the
present study, as only 7 batches were infected, which is a much lower prevalence
compared to the study of Martinez et al. (2011). The latter study detected 8 out of 10
farms positive for the presence of Y. pseudotuberculosis. Studies carried out in other
regions (Estonia, Latvia and Lithuania), showed a higher number of infected batches,
varying from 13 to 60% positive batches (Martinez et al., 2009; Terentjeva and Bezins,
2010; Novoslavskij et al., 2012). In the present study, only 23 out of 7047 pigs (0.3%)
carried Y. pseudotuberculosis. This is a similar result like in most other European countries
as Spain (0%), Italy (1%), Estonia (1%), Latvia (3-5%), Finland (4%) and as reported in Russia
CHAPTER 2 ________________________________________________________________________
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73
(7%) (Martinez et al., 2009; Terentjeva and Bezins, 2010; Martinez et al., 2011). This
variance could be explained by the differences in management system (organic versus
conventional) like mentioned earlier.
The two slaughterhouses showed no significant variation in their results, so the different
input of pigs has no influence (Table VIII). Fredriksson-Ahomaa et al. (2000), who used
different isolation and PCR methods, found a significant difference between
slaughterhouses, which may be due to differences in slaughtering procedures, hygiene
measurements and origin of incoming pigs.
Only few data are available about the number of Y. enterocolitica and Y.
pseudotuberculosis in slaughter pig tonsils at pig- and batch-level (Van Damme et al.,
2010; Van Damme et al., 2012; Chapter 1). Tonsils of 328 pigs contained more than 5.00
log10 Y. enterocolitica g-1 tissue. Highly contaminated pigs may be more likely to cause
cross-contamination during slaughter. To our knowledge, the present study was the first
comprehensive study enumerating Y. pseudotuberculosis in pig tonsils. In the case tonsils
were positive for Y. pseudotuberculosis comparable numbers were present as for Y.
enterocolitica.
In the present study, only Y. enterocolitica serotype O:3 was detected. The dominance in
Belgium of bioserotype 4/O:3 has also been described in Van Damme (2013) and in
Chapter 1. However, Martinez et al. (2011) found bioserotype 3/O:9 in 8 (9%) of the 89
positive samples collected in Belgium. Martinez et al. (2011) did not mention if all
bioserotype 3/O:9 were isolated from pigs originating from the same batch or the same
farm. The latter data indicates that other bioserotypes than 4/O:3 are presented in the
Belgian pig population.
To our knowledge, it is the first time that the presence of the enzyme urease was
considered in isolates derived from pigs on a large scale. This has an implication for the
first screening of potential colonies (Devenish and Schiemann, 1981): to avoid false
negatives, also urease negative colonies should be retained for further testing.
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74
Almost 23% of the isolates were virF-negative, which is similar compared to the study of
Martinez et al. (2009, 2011) and Van Damme et al. (2013) where 19%, 23% and 18.5% of
the isolates from tonsil samples did not carry the pYV respectively. The wide range of
presence of virF-negative samples within a batch raises the question whether loosing the
pYV during isolation is the only cause of pYV-negative human pathogenic Y. enterocolitica
(Van Damme et al., 2013). It can be argued that the inability to produce urease and the
absence of pYV are related, but this is not correlated (data not shown). There is even no
batch included in this study that was completely virF- and urease-negative. Similar to the
results of Martinez et al., (2009), the amount of Y. enterocolitica virF-negative samples is
larger than the Y. pseudotuberculosis virF-negative samples.
In conclusion, there can be a large variation in the within-batch prevalence of human
pathogenic Y. enterocolitica and Y. pseudotuberculosis in pig batches at time of slaughter.
The proposal of the biohazard panel of EFSA is to collect data about the infection rate of
pig batches prior to slaughter (Biohaz, 2011). Due to the large amount of positive batches,
a risk based classification of pig batches before slaughter, as also proposed by EFSA, will
be challenging. Nevertheless, this variation may be used to determine factors which are
influencing this prevalence.
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75
CHAPTER 3
SEROPREVALENCE OF ENTEROPATHOGENIC
YERSINIA SPP. IN PIG BATCHES AT SLAUGHTER
Modified from: Vanantwerpen, G., Van Damme, I., De Zutter, L, Houf, K.., 2014.
Seroprevalence of Enteropathogenic Yersinia spp. in Pig Batches at Slaughter. Preventive
Veterinary Medicine 116, 193-196.
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76
1. Abstract
Enteropathogenic Yersinia spp. are one of the main causes of foodborne bacterial
infections in Europe. Slaughter pigs are the main reservoir and carcasses are
contaminated during a sub-optimal hygienically slaughtering-process. Serology is
potentially an easy option to test for the Yersinia-status of the pig (batches) before
slaughter. A study of the variation in activity values (OD%) of Yersinia spp. in pigs and pig
batches when applying a serological test was therefore conducted. In this study, pieces of
the diaphragm of 7047 pigs, originating from 100 farms, were collected and meat juice
was gathered, where after an enzyme-linked immunosorbent assay (ELISA) Pigtype
Yopscreen (Labor Diagnostik Leipzig, Qiagen, Leipzig, Germany) was performed. The
results were defined positive if the activity values exceeded the proposed cut-off value of
30 OD%. Results at pig level displayed a bimodal-shaped distribution with modes at 0 to
10% (n=879) and 50 to 60% (n=667). The average OD% was 51% and 66% of the animals
tested positive. The within-batch seroprevalence ranged from 0 to 100% and also showed
a bimodal distribution with modes at 0% (n=7) and 85-90% (n=16). On 7 farms, no single
seropositive animal was present and in 22 farms, the mean OD% was below 30%. Based
on the results obtained at slaughter, 66% of the pigs had contact with enteropathogenic
Yersinia spp. at farm level. The latter occurred in at least 93% of the farms. Many farms
are harboring enteropathogenic Yersinia spp.
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2. Introduction
Enteropathogenic Yersinia spp. are an important cause of foodborne bacterial infections
in Europe. Two species, human pathogenic Y. enterocolitica and Y. pseudotuberculosis,
caused 7017 infections or 1.63 infections/100000 inhabitants in 2011 in Europe, with
human pathogenic Y. enterocolitica responsible for more than 98% of these infections
(EFSA and ECDC, 2013). Symptoms range from mild, self-limiting diarrhea to mesenteric
lymphadenitis. However, other chronic disorders like reactive arthritis or erythema
nodosum can also emerge (Bottone, 1997; EFSA and ECDC, 2013). Many animal species
may be carrier of these pathogens, but pigs are regarded as the main reservoir. Handling
and consumption of raw or undercooked pork are the primary risk factors for human
infection (Tauxe et al., 1987; Nikolova et al., 2001; Boqvist et al., 2009; EFSA and ECDC,
2013).
Yersinia strains harboring the virulence plasmid (pYV) are pathogenic for humans. Strains
lacking the pYV like Y. enterocolitica biotype 1A are considered apathogenic (Revell and
Miller, 2001). Pathogenic strains trigger an antibody response against for example an
integrated antihost system encoded on the pYV, the Yop (Yersinia outer membrane
proteins) virulon, which consists of (1) a delivery apparatus and (2) a specialized type III
Yop secretion system (Ysc) allowing the delivery of (3) effector Yops into eukaryotic cells,
the latter controlled by YopN. The effector Yops have different functions: they can disrupt
the cytoskeleton (YopE), round up cells (YopE, YopO) and induce apoptosis of
macrophages (YopP, YopJ) (Rosqvist et al., 1991; Cornelis and Wolf-Watz, 1997; Mills et
al., 1997; Cornelis et al., 1998; Heesemann et al., 2006).
On-farm infections are still active when pigs are delivered to the slaughterhouse. Due to
slaughter procedures, bacteria present in faeces and in the throat of infected pigs can
contaminate other carcasses during processing (Laukkanen et al., 2009). In a recent
qualitative risk analysis of foodborne hazards in pork, human pathogenic Y. enterocolitica
is indicated as a risk of medium relevance for human health in the EU, based on the
probability of occurrence, the severity of consequences and the proportion of cases
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caused by pig meat (Biohaz, 2011). One of the recommendations was to modernize
current meat inspection towards a more risk-based approach. For this reason, it would be
useful to have information about the infection status of slaughter pig batches before their
arrival at the slaughterhouse. After comparing bacteriology and serology, serological data
should allow the identification of high and low risk farms, which creates the opportunity
to implement a risk based porcine meat inspection. Currently, it remains difficult to
determine the infection status of fattening pigs on farms because microbiological isolation
before slaughter requires sampling of the tonsils. Sampling of the faeces is not informative
at moment of slaughter, the pigs may still carry Y. enterocolitica in the tonsils, but not
shed them in the faeces (Nesbakken, 2006). Moreover, since microbiological isolation is
time consuming, serology of meat juice samples from pigs during slaughter may be used
to determine the infection rate of farms. By using serology, it should be possible on a later
stage to link these results with the on-farm infections. Therefore, the aim of this study
was to evaluate if there is variation in the serological prevalence of human pathogenic Y.
enterocolitica in pigs at slaughter.
3. Material and methods
The pigs sampled for the study of Chapter 2 are the same pigs sampled for this study. A
piece of the diaphragm pillar (± 10g) was collected immediately after splitting the carcass.
Upon arrival at the laboratory, all samples were stored at -20°C. After two to three weeks,
the samples were thawed during 48 h at 4°C and 2 ml of meat juice was collected and
stored at -20°C until analysis. The enzyme-linked immunosorbent assay (ELISA) Pigtype
Yopscreen (Labor Diagnostik Leipzig, Qiagen, Leipzig, Germany) was performed according
to the manufacturer’s instructions. The amount of antibodies against Yops, expressed by
the optical density (OD), was determined with a spectrophotometer (Tecan SpectraFluor,
MTX Lab Systems, Virginia, U.S.) at 450 nm. Activity values (OD%) were calculated based
on the measured OD values, and the mean OD values of positive and negative controls
using the following formula:
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79
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑣𝑎𝑙𝑢𝑒 (𝑂𝐷%) = 𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷𝑛𝑒𝑔
𝑂𝐷𝑝𝑜𝑠 − 𝑂𝐷𝑛𝑒𝑔
The results were considered positive if the activity value exceeded the cut-off value of 30
OD% as proposed by the manufacturer. The within-batch seroprevalence is the
comparison of the number of positive pigs to the total number of sampled pigs (1/0 cut-
off 30 OD%). A batch was classified as positive when at least one pig within the batch had
an activity value above the cut-off value.
4. Results
Pigs from 100 batches were examined in two Belgian slaughterhouses. The batch size
varied from 70 to 930 pigs, with a mean batch size of 314 ± 169 pigs. According to previous
calculations, the sample size varied from 41 to 88 pigs per batch (mean: 70 ± 10). In total,
pieces of the diaphragm of 7047 pigs were collected.
The OD% of the individual pigs showed a large variation ranging from -4.9 to 181.6 OD%
(Fig. VIII). The results displayed a bimodal-shaped distribution with modes at 0-10%
(n=879) and 50-60% (n=667). The average OD% was 51%. Sixty-six percent of the animals
were classified as positive according to the used cut-off value. The mean OD% per batch
was also very variable (from -1 to 95). In 22 batches the mean OD% was less than 30%
(Fig. IX).
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The within-batch seroprevalence (pigs with OD%>30% / total pigs sampled per batch)
ranged from 0 to 100% and displayed a bimodal distribution with modes at 0% (n=7) and
85-90% (n=16) (Fig. X). On only seven farms, no seropositive animal was present. The
mean within-batch seroprevalence was 66.4%.
0
100
200
300
400
500
600
700
800
900
1000
num
ber
of
pig
s
activity value (OD%)
0
2
4
6
8
10
12
14
16
num
ber
of
pig
bat
ches
mean activity value (OD%) per batch
Figure VIII. Frequency distribution of activity values (OD%) in meat juice originating from
pigs at slaughter (dashed line: cut-off value of 30 OD%).
Figure IX. Frequency distribution of activity values (OD%) in meat juice of batches (n =
100) of slaughter pigs (dashed line: cut-off 30 OD%).
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5. Discussion
In the present study, the seroprevalence of 66% of pigs at slaughter pointed towards a
frequent contact with enteropathogenic Yersinia spp. during the rearing period. Few
studies reported on the overall prevalence. In the study of Thibodeau et al. (2001)
performed in Canada, an overall prevalence of 66% in 291 tested animals was presented,
using a lipopolysaccharide (LPS)-ELISA. In the study of von Altrock et al. (2011) 80 batches
(30 pigs/batch) were tested, with an overall prevalence of 64.1%. This study was
performed in Germany (Lower Saxony) with the same ELISA kit as the present study. These
results are similar with the one found in the present study.
Few studies are currently available about the within-batch seroprevalence. A research of
Skjerve et al. (1998), conducted in Norway using a LPS-ELISA, sampled 287 batches (5 to
0
2
4
6
8
10
12
14
16
num
ber
of
pig
bat
ches
within-batch seroprevalence (%)
Figure X. Frequency distribution of the within-batch seroprevalence of batches (n=100) of
pigs at slaughter.
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43 pigs/batch), from which 182 (63.5%) were found positive, though the within-batch
prevalence ranged from 0 to 100%. Meemken and Blaha (2011) studied the Yersinia
within-herd seroprevalence in 6 herds, which also ranged from 0 to 100% (10 to 108
pigs/herd), using the same ELISA kit as the present study. In the research of von Altrock
et al. (2011) the average within-herd seroprevalence was 65.7% which is similar to the
present findings. They found 16% of the batches seronegative in contrast to 7% in the
present study. This lower number of seronegative batches could be due to the higher
number of pigs that were sampled per batch.
As shown in Figure IX, many batches show a high mean OD%, which is correlated with a
high level of antibodies in the meat juice. This was also the case with the results found by
von Altrock et al. (2011). However, the number of batches with a seroprevalence (1/0 cut-
off 30 OD%) above the 90% was much higher (52.2%) in the study of von Altrock et al.
(2011) in contrast to the present study (28%). Nevertheless, 44% of the farms in the
present study have a seroprevalence above the 85%, which indicates that positive farms
often deliver a high number of seropositive slaughter pigs.
In line with the EFSA proposal to implement a more risk based porcine meat inspection
(Biohaz, 2011), a categorization of the infection status of batches arriving at the
slaughterhouse should be made. This classification can be either based on microbiological
examination or on serological data. The prevalence based on microbiological analysis of
the tonsils can only be determined pain and stress free and is easy to perform when
samples are taken at slaughterhouse level. Therefore, the prevalence based on serological
analysis of meat juice should be preferred: it could be determined prior to slaughter,
obtaining blood samples from living pigs is less stressful for these pigs compared to
collecting tonsil samples and it can be combined with serological tests for other
pathogens. Nesbakken et al. (2006) stated that serological testing of pigs from 100 days
of age until slaughter age can be applied as a basis for classification between herds free
from Yersinia spp. and infected herds. In the present study, meat juice was selected
instead of blood serum samples as Meemken and Blaha (2011) showed an excellent
agreement between blood serum collected at slaughter age and meat juice.
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A disadvantage of using serology is the possibility that pigs can already be infected but do
not have antibodies yet. It takes 12 to 19 days for seroconversion by experimental
infection -which could be longer in practice-, but only a few hours for contamination of
the tonsils in five-week old piglets by oral inoculation (Nielsen et al., 1996; Thibodeau et
al., 1999). Moreover, one has to take into account that when the infection is finished and
the pigs do not harbor Yersinia spp. any longer, antibodies are still present. The
association between the within-batch microbiological (based on tonsils) and serological
(based on meat juice) prevalence is still not clear. Research about this relationship at time
of slaughter should be considered.
The seroprevalence of pigs at slaughter varies widely between pig batches of different
farms. Due to this large variety, the seroprevalence offers the opportunity to determine
factors that influence this prevalence. Further research about risk factors that determine
the within-batch prevalence should be performed.
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85
CHAPTER 4
PREDICTION OF THE INFECTION STATUS OF PIGS
AND PIG BATCHES AT SLAUGHTER WITH HUMAN
PAHTOGENIC YERSINIA SPP. BASED ON
SEROLOGICAL DATA
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1. Abstract
Pigs are the main reservoir of Y. enterocolitica, and the microbiological and serological
prevalence of this pathogen differs between farms. The infection status of pig batches
arriving at the slaughterhouse is largely unknown. Moreover, a link between the presence
of human pathogenic Yersinia spp. and the presence of antibodies is missing. A relation
between the microbiological and serological prevalence could help to predict the infection
of the pigs prior to slaughter. Pigs from 100 different batches were sampled. Tonsils and
pieces of diaphragm were collected from 7047 pigs (on average 70 pigs per batch). The
tonsils were analyzed using a direct plating method and confirmed with a multiplex
Polymerase Chain Reaction (ail, yst, virF). The meat juice of the diaphragm pillars was
analyzed by Enzyme Linked ImmunoSorbent Assay Pigtype Yopscreen (Labor Diagnostik
Leipzig, Qiagen, Leipzig, Germany). The bacteriological and serological results were
compared using a mixed-effects logistic regression at the pig and the batch level. Yersinia
spp. were found in 2009 pigs, of which 1872 also had antibodies against Yersinia spp.
According to the logistic regression, the microbiological contamination could not be
predicted by the presence of antibodies at pig level. At the batch level, a relation was
observed. A mean activity value of 37 Optical Density (OD)% indicates a microbiological
positive farm. The equation could predict whether a pig batch will include infected pigs
before it arrives at the slaughterhouse.
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2. Introduction
Pigs are identified as the main reservoir of human pathogenic Yersinia enterocolitica
(Tauxe et al., 1987). These Yersinia spp. represent 98.4% of the 7000 confirmed human
yersiniosis cases in the European Union each year, and most of the remaining cases (1.6%)
is caused by Y. pseudotuberculosis (EFSA and ECDC, 2013). Human pathogenic Y.
enterocolitica is also responsible for more than 14000 cases in young children in the
United States each year (Scallan et al., 2013).
Pigs infected at farm level are the main source for the (cross-)contamination of pig
carcasses at the slaughterhouse (Laukkanen et al., 2009). The knowledge of the infection
status of pig batches slaughter may allow a distinction of non-infected and infected pig
batches. This way, the status of pigs and pig batches could be taken into account for the
slaughter ranking in order to decrease the number of contaminated carcasses. A relation
between the presence of Yersinia spp. in pigs at the moment of slaughter and the
presence of antibodies, which can be obtained prior to slaughter, is therefore needed.
There are two common methods to assess the prevalence of human pathogenic Y.
enterocolitica and Y. pseudotuberculosis in pigs and pig batches at slaughter:
microbiological analysis of tonsillar tissue or faeces, or serological analysis of meat juice
or blood. Pigs at slaughter age are not shedding Y. enterocolitica frequently, while their
tonsils are still positive and contain a higher number of Yersinia spp. (Nesbakken et al.,
2006; Van Damme, 2013). Meat juice and blood have a proven excellent agreement
(Meemken and Blaha, 2011).The prevalence obtained by both methods in pigs at
slaughter display a great variation between pig farms (Fukushima et al., 1983; Letellier et
al., 1999; Gurtler et al., 2005; Laukkanen et al., 2009; Novoslavkij et al., 2011). The
microbiological and the serological method also show some discrepancies. Depending on
the age of the pigs to be sampled and the time of primary infection, a choice of method
has to be made. Primarily, the presence of human pathogenic Yersinia spp. in pigs and the
production of antibodies does not follow the same timeframe. A study performed by
Nielsen et al., (1996) followed-up the evolution of antibodies and bacteriology in
experimentally infected pigs. Culture-positive faeces were obtained from day 5 to 21 post
CHAPTER 4 ________________________________________________________________________
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88
infection (p.i.), where after no pigs were shedding Yersinia at day 68 p.i. All inoculated
pigs had seroconverted at day 19 p.i. and remained seropositive until day 70 p.i.
Nesbakken et al. (2006) and Vilar et al. (2013) studied the natural dynamic of infection.
Both studies found a rising antibody-titer starting at an age of 100 days till 5 months.
Nesbakken et al. (2006) indicated the bacteriological examination of faeces useful
between 85 and 135 days of age, while, according to Vilar et al. (2013), the Yersinia-
excretion was peaking in pigs of 2-3 months old. Other studies performed on the natural
dynamics of infection also show a peak in excretion, however at an older age (12-21
weeks) (Fukushima et al., 1983; Gurtler et al., 2005; Virtanen et al., 2012). These studies
indicate that shedding Y. enterocolitica in the faeces happens before antibodies are
produced, resulting in an earlier detection of infection by using the microbiological
method than serology (Fukushima et al., 1983; Nesbakken et al., 2006; Nielsen et al.,
1996). The evolution of antibodies and bacteriology in pigs concerning Y.
pseudotuberculosis was never studied. Secondly, the dilemma of analyzing tonsils or
faeces is also depended on the time of infection. The carriage of enteropathogenic Y.
enterocolitica last several months in the tonsils, whereas faecal excretion decrease within
a few weeks p.i. (Fukushima et al., 1983; Fukushima et al., 1984a; Fukushima et al., 1984b;
Nielsen et al., 1996; Nesbakken et al., 2006; Virtanen et al., 2012). The difference between
tonsillar and faecal sampling is not obvious for Y. pseudotuberculosis (Laukkanen et al.,
2008).
The aim of this study is to provide a predictive value based on serology for the prognosis
of the microbiological status of pigs and pig batches at slaughter age.
3. Material and methods
3.1. Sample collection, microbiological analysis and serological analysis
Sample collection and the microbiological and serological analyses have been already
described in Chapters 2 and 3.
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3.2. Statistical analysis
Three different cut-off values were considered in the interpretation of the serological
results: 25 OD%, 30 OD% and 35 OD%. The cut-off of 30 OD% is the one proposed by the
manufacturer. The value of 25 OD% was previously recommended by the same
manufacturer and because this was 5 OD% lower, also a 5 OD% higher value was taken
into account. Values lower than the cut-off value were considered as negative, values
equal to or higher than the cut-off value were considered as positive. A batch was
considered as positive when at least one sample -tonsil or meat juice- was positive. The
results of both prevalence-determining methods were compared using a mixed-effects
logistic regression at pig and batch level by using Stata/MP 12.1 (StataCorp, 2011). The
freedom of disease as well as the sensitivity and the the specificity of the test with the
microbiology as golden standard was calculated by Win Episcope for both batch and
individual level (Thrusfield et al., 2011). The positive and negative predictive values
depend on the prevalence. They were also calculated using Win Episcope. The normality
tests (Kolmogorov-Smirnov and Shapior-Wilk test) were calculated by SPSS version 21
(IBM, Armonk, New York, U.S.).
4. Results
Yersinia enterocolitica was present in 2009 pigs and 23 animals harbored Y.
pseudotuberculosis. In one pig, both Y. enterocolitica and Y. pseudotuberculosis were
recovered, which makes the total Yersinia spp. infected pigs to 2031 (Chapter 2). Of these
pigs, 1872 also had a level of antibodies against Yersinia spp. above the proposed cut-off
of 30 OD% (Table X). In total, there were 4851 pigs positive in at least one sample matrix.
CHAPTER 4 ________________________________________________________________________
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90
Table X. The categorization of slaughter pigs (n=7047) depending on their microbiological
and serological status.
Antibodies in meat juice
Human pathogenic Yersinia spp. in tonsils Total
Absent Present
Absent* 2196 159 2355 Present* 2820 1872 4692
Total 5016 2031 7047 *based on the activity value using a cut-off of 30 OD%
Out of the 5016 pigs without Yersinia spp. in their tonsils, 2196 had no antibodies against
these bacteria and 2820 pigs possessed the antibodies. The bacteriological negative pigs
displayed a large variation in activity value (-4.90 – 169.08 OD%). However, the majority
of the bacteriological negative pigs (n=5016) yielded a low activity value (Fig. XI). The
number of pigs with an activity value between -5 and 5 OD% is 1105. Most pigs classified
as bacteriological positive (n=2031) also have positive antibody titers (Fig. XII). The results
of the microbiological positive pigs are normally distributed (mean=72.14; standard
deviation=31.80): the result of the Kolmogorov-Smirnov test was 0.033 (P<0.005) and the
result of the Shapiro-Wilk test was 0.994 (P<0.005). Applying the logistic regression, the
microbiological contamination could not be predicted by the presence of antibodies at
the pig level (Fig. XIII). At the pig level, the sensitivity of the test is 92.2% and the specificity
43.8% when the cut-off value is 30 OD% (Table XI). When this cut-off is changed to 25
OD%, the sensitivity slightly increased to 94.2% and the specificity decreased to 40.0%. If
the cut-off is changed to 35 OD%, the sensitivity drops to 88.2%, the specificity increases
to 47.6%.
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91
0
100
200
300
400
500
600
num
ber
of
pig
s
activity value (OD%)
0
20
40
60
80
100
120
140
160
num
ber
of
pig
s
activity value (OD%)
Figure XI. Distribution of the individual pig activity values of microbiological negative
pigs (n=5016).
Figure XII. Distribution of the individual pig activity values of microbiological
positive pigs (n=2031).
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92
Table XI. The sensitivity, specificity, positive and negative predictive value at different cut-
off values (25, 30 and 35 OD%) at pig and batch level with microbiology in tonsils as golden
standard.
25 OD% 30 OD% 35 OD%
Pig level (%)
Batch level (%)
Pig level (%)
Batch level (%)
Pig level (%)
Batch level (%)
Sensitivity 94.2 93.1 92.2 89.7 88.2 89.7 Specificity 40.0 100 43.8 100 47.6 100 Positive predictive value
39.5 100 39.9 100 41.2 100
Negative predictive value
94.3 68.4 93.2 59.1 90.7 59.1
Figure XIII. Scatter plot of the concentration of Yersinia spp. in the tonsils and the
activity value of the antibodies against Yersinia spp. in meat juice collected from
diaphragm at pig level.
-50
.0
0
5
0.0
1
00
.0
15
0.0
2
00
.0
acti
vity
val
ue
(OD
%)
0 1.00 2.00 3.00 4.00 5.00 6.00
Yersinia count (log10 CFU/g tonsil)
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93
At batch level, 87 batches were infected with Yersinia spp. from which 78 batches had a
mean within-batch OD% above 30 OD% (Table XII). There were 87 batches that had at
least one pig positive in one sample matrix. None of the negative batches for Yersinia spp.
in the tonsils had activity values of more than 30 OD% in the meat juice. At batch level,
the sensitivity of the serology test (30 OD%) was 89.7%, while the specificity was
calculated at 100%. These values remain the same when the cut-off value is changed to
35 OD%. However, the sensitivity increases when the cut-off value is altered to 25 OD%.
The mixed-effects logistic regression resulted in the following formula:
𝑤𝑖𝑡ℎ𝑖𝑛 − 𝑏𝑎𝑡𝑐ℎ 𝑚𝑖𝑐𝑟𝑜𝑏𝑖𝑜𝑙𝑜𝑔𝑖𝑐𝑎𝑙 𝑝𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 = 0.444
1 − 𝑒−0.063∗(𝑚𝑒𝑎𝑛 𝑂𝐷%−37.069)
for which the cut-off value for a positive farm is 37 OD% (Fig. XIV).
Table XII. The categorization of batches of slaughter pigs (n=100) depending on their
microbiological and serological status.
Antibodies in meat juice
Human pathogenic Yersinia spp. in tonsils Total
Absent Present
Absent* 13 9 22 Present* 0 78 78
Total 13 87 100 * based on the activity value using a cut-off of 30OD%
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94
5. Discussion
Since pork is a main source for human yersiniosis, it is important to reduce the prevalence
of Yersinia spp. in pork as much as possible. A main strategy of the reduction of the risk of
pig carcass contamination is the decrease of the number of infected slaughter pigs
(Laukkanen et al., 2009). In Europe, there is no control program available or in use to
decrease this on-farm prevalence yet. Another possibility is to reduce the contamination
during slaughter by bagging the rectum (Nesbakken et al., 1984; Laukkanen et al., 2010b)
The presence of Yersinia spp. in a batch is important regarding contamination of carcasses
and is the basis when logistic slaughtering is applied. Therefore, knowledge of the
infection status is needed before pigs are slaughtered. The comparison between
microbiology and serology for Yersinia spp. at pig and batch level only results in a relation
0 20 40 60 80 100
mean within-batch activity value (OD%)
0
2
0
40
60
wit
hin
-bat
ch b
acte
rio
logi
cal p
reva
len
ce (
%)
Figure XIV. Scatter plot of the mean within-batch activity value of the antibodies against
Yersinia spp. in meat juice collected from diaphragm and the within-batch
bacteriological prevalence of Yersinia spp. in the tonsils at batch level.
CHAPTER 4 ________________________________________________________________________
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95
at batch level. Overall, a weak agreement was found between bacteriology and serology
for Yersinia spp. diagnosis. This is in line with Nollet et al. (2005) who compared the
presence of Salmonella spp. and of antibodies against Salmonella spp. in slaughter pigs.
There was also a weak agreement between these two diagnostic procedures.
Pigs without human pathogenic Yersinia spp. in their tonsils, most (56.2%) are
serologically classified as positive due to an activity value above the cut-off value of 30
OD%. So the proposed cut-off of 30 OD% is not reliable to detect microbiological negative
pigs. Decreasing the cut-off activity value (e.g. 25 OD%) leads to a higher detection of
infected pigs (sensitivity is 94.2%), which is the most important. However, this also leads
to indicating more non-infected pigs as infected. Microbiological negative pigs can present
a wide range of activity values. An already cleared infection could be an explanation for
the presence of these antibodies. Nielsen et al. (1996) showed that antibodies are still
present 70 days p.i. (end of the experiment) while excretion was finished at 68 days p.i.,
which is comparable with the study of Vilar et al. (2013), where antibodies were still
present 5 months p.i., while excretion was finished after 2-3 months p.i. To our
knowledge, a(n) (experimental) study of the evolution of Yersinia spp. in the tonsils
instead of faecal excretion and serology do not exist.
A very low amount of the microbiological positive pigs (7.8%) presented an activity value
below the proposed cut-off of 30 OD%. An explanation for the discrepancy is the biological
difference between the presence of the pathogen and the serological reaction in the
animal. Presence of the Yersinia spp. in the tonsils is due to a recent infection, whereby
no antibodies will be detected in the serum of these infected animals. According to
Thibodeau et al. (1999), tonsils can already be colonized a few hours post infection, while
infected pigs are all seroconverted around 19 days p.i. (Nielsen et al., 1996).
Based on serology results it is possible to detect infected batches prior to slaughter by
using the presently proposed equation: when the mean activity value is more than 37
OD%, the batch is indicated positive. Knowing the microbiological prevalence prior to
slaughter, infected pig batches can be delivered to the abattoir and slaughtered after non-
CHAPTER 4 ________________________________________________________________________
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96
infected pig batches to avoid cross-contamination in the slaughterhouse. To prove
freedom of disease, from a batch of 50 pigs, 12 pigs should be sampled (based on a mean
within-batch bacteriologic prevalence of 30%). When the batch counts more pigs, 13 pigs
should be sampled. It is more important to detect microbiological positive batches than
pigs, since separating infected and non-infected pigs at the slaughterhouse is impossible.
If pig batches are categorized in the slaughterhouse based on serological testing, it should
be taken into account that slaughter pigs can still harbor Yersinia spp. in the tonsils,
without seroconversion (9 of the 87 batches). The sensitivity of the serological test at
batch level was 89.7% using a cut-off of 30 OD%. The use of the lowest cut-off value (25
OD%) is recommended in order to increase the probability of classifying positive herds
correctly based on serological testing. However, the lower the cut-off value, the higher
the number of seropositive batches detected although the animals are not harboring the
bacteria (the highest mean activity value of a microbiological negative batch was 20.6
OD%). Though Yersinia spp. are no longer present in those kind of batches, a positive
serological result at the batch level means that Yersinia is or has been present in the batch
and the farmer needs to lower this prevalence. As shown in Fig. XIV, batches with a low
mean activity value, were also infected at a low level. This is fundamental when dividing
batches as low or high infected. To the contrary, low infected batches could have a high
mean activity value.
To decrease the infection in a batch, the number of piglet suppliers can be decreased, the
presence of other pig farms in the area should be as low as possible, semi slatted floors in
the fattening pig unit should be available (Chapter 5). Logistic slaughtering has been also
proven to be useful in blocking the spread and (cross)-contamination of Salmonella spp.
on pig carcasses during slaughter (Swanenburg et al., 2001; Arguello et al., 2014). Both
studies indicate that, next to an accurate batch separation according to their
seroprevalence levels, strict measures for cleaning and disinfection in the lairage and the
slaughterhouse facilities are needed when logistic slaughter is performed. Due to a
decreasing prevalence of Salmonella at herd level, infections at transport and lairage
became more and more important. A separation based on the herd status and executed
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while transporting the pigs, significantly decreased the percentage of infected pigs that
became infected at lairage (Hotes et al., 2011).
In conclusion, only a weak agreement was found between the results of both methods at
individual level. Serological screening methods could be useful at batch level to distinguish
infected from non-infected batches. In order to correctly classify batches, 13 samples per
batch (batches larger than 100 pigs) should be sampled. Serological testing of pigs prior
to slaughter followed by classifying the batches (infected and non-infected) and adapting
the slaughter order could be useful to decrease the risk of contamination during
slaughtering procedures.
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CHAPTER 5
ASSESSMENT OF RISK FACTORS FOR A HIGH
WITHIN-BATCH PREVALENCE OF YERSINIA
ENTEROCOLITICA IN PIGS BASED ON
MICROBIOLOGICAL ANALYSIS AT SLAUGHTER
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1. Abstract
The purpose of the current study was to find factors at farm-level influencing the
bacteriological prevalence of Y. enterocolitica in pigs at time of slaughter. On 100 farms,
data concerning housing, ventilation, biosecurity, management, feeding and disease
control were collected using a face-to-face questionnaire. At the slaughterhouse, tonsils
of on average 70 slaughter pigs per batch were sampled to determine the infection status
of pigs. After univariate mixed effect logistic regressions, variables which were related to
the Yersinia prevalence (P < 0.05) were included in a multivariate model. In this model,
the factors remaining positively associated with a higher Y. enterocolitica carriage in the
tonsils (P<0.1) were an increasing number of piglet suppliers, a high density of pig farms
in the area and the use of semi slatted floors in the fattening pig unit. The proper use of a
disinfection bath before entering the stables and a poor biosecurity level were protective
factors, although a higher prevalence was associated with a significant positive interaction
between the presence of pets in the stables and a poor biosecurity level. Reducing the
number of piglet suppliers and using a disinfection bath properly could be easily
implemented by pig farmers to lower the prevalence of Y. enterocolitica in pigs at
slaughter.
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2. Introduction
Yersiniosis is an important food-borne disease responsible for about 7000 confirmed
human cases per year in the European Union and around 14600 infections in young
children in the U.S. each year (EFSA and ECDC, 2013, Scallan et al., 2013). Infections are
mainly caused by the human pathogenic biotypes of Yersinia enterocolitica (98.4%) and
to a lesser extent, Y. pseudotuberculosis (0.9%). Pork and products thereof are considered
as the most important infection source. Contamination of the meat occurs mainly by
(cross-)contamination during slaughter by pigs harboring Yersiniae in their tonsils or by
faecal shedding (Laukkanen et al., 2009). Since pigs have been identified as the main
reservoir of these pathogens, the European Food Safety Authority (EFSA) has published in
a recent scientific opinion (Biohaz, 2011) to assist the reduction of the contamination risk
in the pig slaughterhouses by suggesting to decrease the number of infected animals at
farm-level. Decreasing the occurrence of Y. enterocolitica in pigs and ultimately on pork
is considered as the most important control step towards reducing the number of human
infections, therefore the infection rate of delivered batches prior to slaughter should be
known so that appropriate actions in the slaughterhouse can be taken.
Studies of risk factors for the occurrence of Y. enterocolitica at pig farm level have been
performed in several countries, but, except for the study of Wesley et al. (2008) in the
U.S., these were always based on a limited number of farms or pigs per farm sampled
(Nowak et al., 2006; Laukkanen et al., 2008; Laukkanen et al., 2009; Virtanen et al., 2012;
Novoslavskij et al., 2013). So-far, common findings are that the use of bedding material,
the purchase of piglets originating from more than one farm, daily observation of the
presence of a cat in the stables, drinking from nipples and snout contact between pigs
from adhering pens in the fattening pig unit are risk factors for infection. The use of
municipal water, a farrow-to-finish approach and organic farming acted as protective
factors (Laukkanen et al., 2009; Virtanen et al., 2012; Novoslavskij et al., 2013; Vilar et al.,
2013). Certain intervention strategies based on these factors, could reduce the within-
batch prevalence prior to slaughter, but have not been implemented so far.
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As transmission already starts at farm-level, several studies have assessed the prevalence
prior to slaughter (Nowak et al., 2006; Laukkanen et al., 2009, Fondrevez et al., 2010;
Virtanen et al., 2011; Novoslavskij et al., 2012). They revealed a large variation in
prevalence between pig batches originating from different farms allowing determination
of factors influencing the infection status.
The aim of the present study is to assess different farm factors influencing the
microbiological prevalence of human pathogenic Y. enterocolitica in pigs at slaughter to
obtain interventions to reduce this prevalence and that finally decrease the level of Y.
enterocolitica (cross-)contamination in the slaughterhouse.
3. Material and methods
3.1. Study design and sample collection
The collection of samples has already been discussed in Chapter 2. The farms were
scattered all over Belgium, with most batches located in West-Flanders related to the high
density of pig farms in this province (more than 50% of all Belgian pig farms).
3.2. Collection of questionnaire data
In the present study, shortly before the pigs were slaughtered, each farm was visited and
a face-to-face questionnaire was filled in combined with a guided tour on the farm. The
Yersinia status of the farm was still unknown and the questioning was always performed
by the same investigator. In total, 59 fattening pig herds and 41 farrow-to-finish herds
were included. The number of sows in the farrow-to-finish farms varied between 80 and
750, and the size of the fattening pig farms ranged from 297 to 10,500 slaughter pigs. The
items in the questionnaire were related to the following aspects: production parameters,
type of farm management and housing system, biosecurity and hygiene measurements,
animal management, origin of the drinking water, type of feed and veterinary support
(Table XIII). In total, sixty-eight questions were asked to the farmer or evaluated during
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the visit. Most of the data were binomial (questions answered by yes or no), some
categorical (e.g. feed: (1) grain, (2) meal, (3) porridge) or continuous (e.g. daily weight
gain). A Specific Pathogen Free (SPF) farm is free from Mycoplasma hyopneumoniae
(enzootic pneumonia), certain serotypes of Actinobacillus pleuropneumoniae, Porcine
Reproductive and Respiratory Syndrome Virus (PRRSV), Sarcoptes scabiei (mange) and
lice. The yearly average Salmonella result, expressed by the ratio of sample to positive
(S/P), was calculated based on farm specific official reports.
3.3. Statistical method
Excel software and Stata/MP 12.1 (StataCorp, 2011) were used for all analyses. The
dependent variable was defined as the infection status of the animals (presence/absence
of Y. enterocolitica). Independent variables included categorical, continuous and binomial
farm-level variables. When the correlation coefficient between farm variables was high (r
≥ |0.7|), only one of the variables was further included in the model. The decision
regarding which variable to include depended on the biological plausibility. In this study,
the number of slaughter pigs present was chosen instead of the number of sows (Table
XIII).
The association between each independent variable and the outcome was first screened
using an univariate mixed effect logistic regression. Variables with P < 0.05 in this
univariate analysis were retained for the multivariate model with farm as random effect.
In this model, only items which remained significantly associated with Y. enterocolitica
carriage in the tonsils (P < 0.1) were determined either as risk or as protective factors. All
possible interactions were evaluated and included when significant (P < 0.1). The mean
within-batch prevalence was calculated based on the number of piglet suppliers and
differences were evaluated using one sample t-tests (P < 0.1).
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Table XIII. List of variables used in the questionnaire and their reason of exclusion from
the analysis.
Characteristic Modality Reason of exclusiona
General information
Batch size
Number of slaughter pigs present
Number of sows present 4
Specific Pathogen Free farm 1
Production parameters
Daily weight gain 3
Mortality ratio
Feed conversion
Management and housing system
Type of farm Farrow-to-finish – fattening pig farm
Floor type in slaughter pig stables
Fully slatted floor – semi-slatted floor
Snout contact possible between pigs from adhering pens
Use of straw bedding in the nursery or fattening pig unit
1
Appropriate temperature change in fattening pig unit
1
Appropriate humidity in the stables
1
Biosecurity and hygiene measurements
all-in/all out system in fattening pig unit
rodents visible in the stable
rodent control program
large amount of flies in the stables
outside the stable: hard and clean ground
pets allowed in the stable
birds present in the fattening unit
grid present in the air openings 1
wild boars present in the neighborhood
1
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Characteristic Modality Reason of exclusiona
cleaning after each rearing round
2 disinfection after each rearing
round
stable stays empty during ≥ 3days after each rearing round
control cleaning and disinfection with hygienogram
1
presence of other pig farms in the area (closer than 500m)
bath placed inside the stable 2
bath content renewed at least every 2 weeks
farm clothes available for visitors 1
other clothes for each compartment
1
presence of a hygiene lock before entering the stables
need to shower before entering the stables
1
use of separate clean and dirty road
use of other cleaning material for each compartment
disinfection material to compel pigs
1
disinfection loading place
farm specific material to fix pigs 1
different shoes for entering the pen with ill pigs
1
farm specific manure tubes
Animal management
number of piglet suppliers
age arriving piglets 1
farm of origin ≥ health statute 3
smaller pigs are relocated in new pens
maximum capacity load fattening pig unit reached
Drinking water
origin of water supply Rain – municipal water
use of acidified water in the nursery
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Characteristic Modality Reason of exclusiona
use of acidified water in the sow unit
1
use of acidified water in the fattening pig unit
1
yearly cleaning and disinfection of the pipes
yearly bacteriologic control of the water
daily control of functioning nipples
Feed
type of feed: fattening pigs Grain – meal - porridge
type of feed: sows Grain – meal - porridge
3
type of feed: piglets Grain – meal - porridge
3
Acidified feed: fattening pigs 3
Acidified feed: sows 3
Acidified feed: piglets
Origin feed Commercial – tailor-made
Type of feed supply Automatic - manual
rodent-free feed storage
Veterinary support
presence of Brachispira spp. 1
presence of Lawsonia intracellularis
1
mean S/P Salmonella ratio last year
past standardized antibiotic treatments
use of anthelmintica in fattening pig unit
End of rearing period
feed stop 12-16 h before loading 1
transport vehicle cleaned and disinfected
3
a1: farms applying the factor: five or less, or 95 or more 2: pooled factors 3: missing values 4: correlation coefficient ≥ ǀ0.7ǀ
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4. Results
When visiting the pig farms, it was noticed that pigs belonging to the same slaughtering
batches could be derived from different stables. The answers to the questions were
sometimes different between these stables (e.g. floor type, type of feed). In this case, the
stable where the majority of the pigs were staying, was chosen to fill in the questionnaire.
After a first evaluation of the 100 questionnaires, some of the collected items turned out
to be unusable due to the low variance between farms, as for instance the use of straw
bedding in the nursery or fattening pig unit (only applied by one of the 100 farms), the
obligated use of a shower for visitors before entering the stables (3%), the use of different
shoes when entering the pen with the ill pigs (1%) and the use of rainwater tanks or wells
as drinking water supply (100%). Therefore, in the complete study, items with five or less
farms applying this item were ruled out, resulting in the exclusion of 19 out of 68 items.
Some close-related items were pooled in the univariate analysis, e.g. the item ‘cleaning-
disinfection-stand empty (‘CDE’) consists of (1) the cleaning and (2) the disinfection of the
stable after each rearing round and (3) the time period in which the stable stood empty
after the pigs were slaughtered (cut-off: ≥ 3 days). A ‘proper use of a disinfection bath’
consists of (1) whether the bath is placed inside the stable, so it is protected from weather
conditions and one is obliged to step in it and (2) the bath is cleaned at least every two
weeks. This limit of two weeks was based on the efficacy data provided by the most
frequently used products (e.g. MS Kiemkill tabs, Schippers, Arendonk, Belgium). Items
with more than 15 missing responses, due to a clueless farmer or person responsible for
the stable, were omitted. As a result, only 36 explanatory items were taken further into
account (Table XIV).
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Table XIV. Description of the remaining binomial and categorical pig farm characteristics
in the 100 visited farms.
Characteristic Modality Number of
farms
Management and housing system
farrow-to-finish production 41
fully slatted floor in fattening pig unit 81
snout contact possible between pigs from adhering pens
67
Biosecurity and hygiene measurements
all-in/all out system in fattening pig unit
80
rodents visible in the stable 37
proper rodent control 86
pets allowed in the stable 43
birds present in the fattening unit 23
poor CDE* no cleaning no disinfection stable empty
during < 3days total poor CDE
30 41 28
45
presence of other pig farms in the area (closer than 500m)
63
correct use of disinfection bath bath inside the stable
bath cleaned at least every 2 weeks
total
37
31
31
farm clothes available for visitors 88
presence of a hygiene lock before entering the stables
49
Animal management
number of piglet suppliers 0 1 2 3 4 >5
41 34 8 5 6 6
Drinking water
use of municipal water 10
use of acidified water in the nursery 19
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Characteristic Modality Number of
farms Feed
type of feed fattening pigs grain meal porridge
7 91 2
commercial feed 89
rodent-free feed storage 93
Veterinary support
past standardized antibiotic treatments 58
use of anthelmintica 94
*CDE: cleaning-disinfection-stand empty
After the univariate analysis, only seven factors were significant (P < 0.05), which were all
included in the multivariate analysis. During this analysis, the element ‘snout contact
possible between pigs from adhering pens’ (P-valueunivariate = 0.045) was excluded due to
a P-valuemultivariate > 0.1. In the final logistic regression model, three risk factors, two
protective factors and one significant interaction were identified (Table XV). Significant
risk factors were ‘the use of a semi-slatted floor in the fattening pig unit’, ‘presence of
other pig farms in the area (closer than 500m)’ and ‘the number of piglet suppliers’ (P <
0.1). Protective factors were ‘the proper use of a disinfection bath’ and a ‘poor CDE’. A
significant interaction (P < 0.1) was observed between a ‘poor CDE’ and ‘the presence of
pets in the stable’, which led to an increasing prevalence.
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Table XV. Logistic regression model with a random effect for farm, of variables
significantly (P ≤ 0.1) associated with the presence of human pathogenic Y. enterocolitica
in Belgian pig batches at slaughter (n = 100 farms).
Factor Odds ratio P-value 95% CI
Semi slatted floor in fattening unit 1.65 0.085 [1.03; 2.64]
Presence of other pig farms in the area (closer than 500m)
1.63 0.036 [1.03; 2.59]
Number of piglet suppliers 1.15 0.019 [1.02; 1.28]
Proper use of disinfection bath 0.58 0.032 [0.36; 0.95]
Poor Cleaning-Disinfection-Stand empty (CDE)
0.50 0.022 [0.28; 0.90]
Interaction: poor CDE and pets 2.39 0.065 [1.05; 5.53]
The mean within-batch prevalence increased with an increasing number of piglet
suppliers (Fig. XV). Nevertheless, there was no significant difference between the mean
within-batch prevalence of farrow-to-finish farms (25.9%; n=41 farms) and fattening pig
farms with only one supplier (25.2%; n=34 farms) (P > 0.1). When more suppliers are
involved, the mean within-batch prevalence increased for two suppliers to 29.7% (eight
farms), for three suppliers to 36.9% (five farms) and for four or more suppliers to 42.5%
(12 farms) (P < 0.1).
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The mean within-batch prevalence of both binary outcomes of the risk and protective
factors was determined (Fig. XVI). The difference between the use of a semi (n=19) and
fully (n=81) slatted floor was stated by a prevalence of 36.0% and 26.7% respectively (P <
0.1). With other pig farms in the area, the prevalence increased from 24.5% (n=37)
towards 30.9% (n=63) (P > 0.1). A third difference between the mean within-batch
prevalence in farms using a disinfection bath properly (n=31) was 20.4% compared to
32.2% in farms without the proper use of such a bath (n=69) (P < 0.1). The impact of the
second protective factor, a poor CDE, was demonstrated by a prevalence of 24.2% for
farms with a poor CDE (n=45) compared to 32.0% in farms which applied good CDE (n=55)
(P < 0.1). When pets were allowed in the stables (n=43), the prevalence was 32.1%
compared to 25.8% when no pets were found in the stables (n=57) (P > 0.1). Comparing
the two latter factors, 31 farms had a poor CDE and no pets in their stables, what resulted
in a mean within-batch prevalence of 20.3% (Fig. XVII). Fourteen farms also had a poor
CDE, but did allow pets in the stables. These farms obtained a prevalence (32.9%) similar
to those farms with good CDE and pets present in the stables (n=29; 31.8%) and the farms
with good CDE and no pets (n=26; 32.2%). When there was a possible snout contact in the
fattening pig unit, the prevalence was higher (33.1%; n=67) than when there was no
possibility to have snout contact (26.2%; n=33).
0
5
10
15
20
25
30
35
40
45
0
(n=41)
1
(n=34)
2
(n=8)
3
(n=5)≥4
(n=12)
mea
n w
ithin
-bat
ch p
reval
ence
(%
)
number of piglet suppliers
Figure XV. The distribution of the mean within-batch prevalence according to farms with
a different number of piglet suppliers (n=100).
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Figure XVI. Comparison of the mean within-batch prevalence between the two possible
outcomes of seven variables.
Figure XVII. Comparison of the mean within-batch prevalence depending on the
presence of poor Cleaning-Disinfection-Stand empty (CDE-) and pets in the stables.
0
5
10
15
20
25
30
35
40
semi slatted
floor
presence of
other pig
farms in the
area
(<500m)
proper use
disinfection
bath
poor CDE pets
allowed in
the stable
possible
snout
contact
mea
n w
ithin
-bat
ch p
reval
ence
(%
)
yes
no
0
5
10
15
20
25
30
35
CDE- pet+
(n=14)
CDE- pet-
(n=31)
CDE+ pet+
(n=29)
CDE+ pet-
(n=26)
mea
n w
ithin
-bat
ch p
reval
ence
(%
)
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5. Discussion
The present study assessed different risk factors based on the presence of human
pathogenic Y. enterocolitica in pig tonsils at time of slaughter originating from 100 farms,
taking into account clustering per batch. As the prevalence based on bacteriological data
at time of slaughter leads to the final carcass contamination (Laukkanen et al., 2009), risk
factors related to this prevalence, are also affecting this contamination.
Based on a much larger number of farms, as well as pigs examined in the present study,
some previous factors could be confirmed, other factors were new or even contradicted
previous studies.
In the present study, factors increasing the within-batch prevalence at time of slaughter
are identified as the presence of semi-slatted floor in the fattening pig unit and the
presence of other pig farms in the neighborhood (<500m). The occurrence of Y.
enterocolitica in a batch is also augmented with the number of piglet suppliers. The proper
use of a disinfection bath and a poor CDE, without pets in the stable, are other factors
that have a decreasing influence on the prevalence.
The first two risk factors, ‘the use of a semi-slatted floor in the fattening pig unit’ and
‘presence of other pig farms in the area (<500m)’ can probably not be remediated
immediately. The use of a semi slatted floor increases the prevalence based on
bacteriological results compared to a fully slatted floor, represented by an 10% increase
in mean within-batch prevalence of 36% and 26.6% respectively. The influence of the type
of floor can be explained by the contact time with pig faeces, in which Y. enterocolitica is
present. Semi-slatted floors create a more constant contact with the pig faeces
(Fukushima et al., 1983; Nielsen et al., 1996; Nesbakken, 2006). The second risk factor
‘presence of other pig farms in the area (<500m)’ was never studied before and indicates
a possible transmission route between nearby farms. The origin of this spread is however
still unknown.
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The third risk factor, ‘the number of piglet suppliers’, differed between farms with none
or one supplier, and farms with more suppliers. The maximum number of piglet suppliers
in this study was 11 per farm. It is known that incoming piglets, potentially infected on
multiplying farms are a possible source for spreading and infection on fattening pig farms
(Virtanen et al., 2012). When piglets arrive from different multiplying farms, there is more
chance to purchase infected, Yersinia-excreting piglets. Purchasing piglets from more than
one farm was also identified as a risk factor in the studies of Nowak et al., (2006) and Vilar
et al., (2013). The present study indicates an equal risk between fattening farms when
purchasing piglets from one supplier and farrow-to-finish farms, which is similar to the
study of Vilar et al. (2013).
In addition, two new protective factors were identified and both are related to hygienic
measurements. The first one, ‘proper use of a disinfection bath’, decreases the spread
between different stables on the same farm, so infections stay more localized. The
difference with the ‘farm clothes available for visitors’, which is not a significant factor, is
that the disinfection bath is also used by the farmer, and not only by the farm visitors. In
this way, infections introduced from outside the farm, and even within the farm but from
different stables, may be eliminated by this disinfection bath. The second protective
factor, a ‘poor CDE’, is more difficult to interpret. Similar results are available for the
presence of Salmonella on pig farms. Van der Wolf et al. (2001) reported that the omission
of disinfection of pig stables was associated with a lower Salmonella seroprevalence
compared to herds that sometimes or always applied disinfection. Moreover, Poljak et al.
(2008) showed that increased frequency of cleaning with cold water and disinfection was
positively correlated with Salmonella shedding. Moreover, when pets are allowed into the
stable, ‘poor CDE’ is a risk factor for a higher prevalence. When pets are allowed in and
move between stables, they keep the infection going as they act as carriers and
transmitters of Y. enterocolitica (Yanagawa et al., 1977; Fredriksson-Ahomaa, 2001;
Murphy et al., 2010). The use of an all-in/all-out management was also identified as
protective factor in a more limited study of Vilar et al. (2013). Novoslavskij et al. (2013)
reported a low biosecurity level as a risk factor. However, in the latter study, the
biosecurity factor included other factors than these in the present study.
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Some factors were not included in the analysis or were not significant in the present study,
but turned out to be risk or protective factors in other studies. The use of a straw bedding
was initially taken into account in the questionnaire, but as applied in only one farm, was
not further included. The use of a bedding was in other studies identified as a risk factor
(Laukkanen et al., 2009; Vilar et al., 2013). Moreover, the use of municipal water is
considered as a protective factor (Virtanen et al., 2012; Vilar et al., 2013), though this
factor was excluded, as all farms used rainwater tanks or wells as drinking water supply.
It is an interesting subject to take into account in future studies. A higher production
capacity has been associated with a higher prevalence of Y. enterocolitica, due to
underlying risk factors inherent in a higher production (Laukkanen et al., 2009; Laukkanen
et al., 2010b). Nevertheless, no differentiation was found in the present study. Feeding
factors have also been related with the prevalence of Y. enterocolitica. Manual feeding of
slaughter pigs has been determined as a protective factor. Use of commercial feed,
presence of meat or bone meal in grower-finisher diet and industrial by-products in feed
have been associated with a higher Y. enterocolitica infection rate. Feed producing
companies can have a positive or negative influence on the prevalence (Nowak et al.,
2006; Wesley et al., 2008; Virtanen et al., 2011). In the present study, no feed related
significant differences were found.
To our knowledge, there are two studies available that performed a risk factors analysis
based on serology (Skjerve et al., 1998; von Altrock et al., 2011). von Altrock et al. (2011)
mentioned the use of a fully slatted floor and the use of municipal water as protective
factors in German pig farms. Skjerve et al. (1998) identified the daily presence of a cat
with kittens in the stable and the use of a bedding as risk factors, while a farrow-to-finish
farm was seen as a protective factor.
In the general picture of pig producing management, another food borne pathogen,
Salmonella, is also very important. The risk and protective factors for a higher prevalence
of Y. enterocolitica on pig farms could be conflicting with factors influencing the
prevalence of Salmonella on pig farms. In a study of Vico et al. (2011) the mesenteric
lymph nodes of pigs were collected in the slaughterhouse and analyzed for the presence
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of Salmonella. Risk factors in this study were lack of rodent control programs, fattening
pig herds, herds managed by more than one full-time worker, municipal water as drinking
water supply and relatively long fattening times. Only one factor is similar between the
study about Salmonella and the present study: pig herds with one or more piglet suppliers
(fattening pig herds) are a risk for a higher prevalence of Y. enterocolitica and Salmonella.
Cardinale et al. (2010) performed a risk factor study based on the bacteriological
Salmonella status of 60 farms by analyzing faecal samples and socks. The prevalence
increased when there was no disinfection at the farrowing stage, when large numbers of
cockroaches were present and when birds were seen in the stable. A lower level of
Salmonella was reached when the technical personnel visited the stable less than once a
month, when castration of piglets was done after 1 week of age and when the all-in all-
out system was respected. Garcia-Feliz et al. (2009) analyzed faecal samples for the
presence of Salmonella. The only two risk factors given in this study were the feeding of
pelleted feed and a high production rate. No factors of the two latter studies were similar
compared to the present study.
In conclusion, reducing the number of piglet suppliers, using a disinfection bath properly
and prohibiting the entrance of pets into the stable are factors that are easily
implemented to lower the prevalence of Y. enterocolitica in pigs at slaughter.
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CHAPTER 6
FACTORS INFLUENCING THE SEROPREVALENCE OF
HUMAN PATHOGENIC YERSINIA SPP. IN
FATTENING PIGS AT SLAUGHTER
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1. Abstract
The main objective of this study was to analyze potential herd-level factors associated
with the detection of Yersinia antibodies in fattening pigs at time of slaughter which gives
the opportunity to create interventions to decrease the presence of these antibodies. The
seroprevalence of Yersinia spp. varies greatly between pig farms. Due to this variation,
risk factors can be determined which may be applied in the pig farm management so the
seroprevalence prior to slaughter will decrease. One hundred farms were visited and
during a face-to-face questionnaire data concerning housing, ventilation, biosecurity,
management, feeding and disease control were collected. At the slaughterhouse, pieces
of diaphragm were collected, where after the meat juice was gathered and an ELISA was
performed to determine the seroprevalence. After using univariate mixed effect logistic
regressions, variables which were related to the Yersinia prevalence (P < 0.05) were
included in a multivariate model. In this model, four risk factors and one protective factor
remained significantly associated with antibodies against Yersinia species in meat juice
(P<0.1). Many piglet suppliers, a high density of pig farms in the area and the use of semi
slatted floors in the fattening pig unit were risk factors. The possibility of snout contact in
the fattening pig unit was a protective factor, although a significant positive interaction
between the presence of pets in the stables and snout contact was observed.
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2. Introduction
Human pathogenic Y. enterocolitica (98.4%) and Y. pseudotuberculosis (0.9%) are causing
the third most important bacterial food-borne disease which leads to about 7000
confirmed human cases in the European Union each year (EFSA and ECDC, 2013). Pigs are
the main reservoir of these pathogens (Thibodeau et al., 1999; Fredriksson-Ahomaa,
2001). The infection is mainly caused by consumption of pork and products thereof.
Contamination of pork occurs by (cross-)contamination during slaughter or following
steps, due to infected pigs (Laukkanen et al., 2009). A reduction of the number of infected
pigs at farm-level would reduce the amount of contaminated carcasses. To know the
number of infected pigs prior to slaughter, there are three possible samples: tonsils,
faeces or blood. Microbiological isolation before slaughter requires sampling of the tonsils
as pigs are intermittent shedders and most pigs no longer shed enteropathogenic Yersinia
spp. in the faeces at slaughter age, thus resulting in an underestimation of the prevalence
when analyzing faecal samples. Nevertheless, sampling of tonsils in living pigs is not
animal-friendly (Fukushima et al., 1983; Thibodeau et al., 1999; Nesbakken, 2006), so, if
sampling happens before slaughter, serological analysis is the most appropriate method.
The number of studies based on the serological prevalence of Yersinia spp. are limited
(Skjerve et al., 1998; von Altrock et al., 2011). The study of Skjerve et al. (1998) was based
only on the antibodies against Y. enterocolitica serotype O:3 and the risk factor analysis
was based on 265 slaughter pig producing farms (conventional and farrow-to-finish
production) and sampled 5 pigs per herd, while the study of von Altrock et al. (2011)
assessed the level of antibodies against both enteropathogenic Yersinia spp., analyzing in
80 herds 30 blood samples per herd. Snout contact, use of tetracycline, fasting pigs before
slaughter, the use of bedding material, daily observation of a cat in the stables and
drinking from nipples were identified as risk factors. Common protective factors were the
use of municipal water, a farrow-to-finish farm and manual feeding of slaughter pigs.
It is important to determine farm factors influencing this seroprevalence, so farmers could
adapt their farm management by introducing these factors to decrease the within-batch
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prevalence. The aim of this study is to gain information about these farm factors to
influence the within-batch seroprevalence of pigs at time of slaughter so that
measurements can be taken to reduce this within-batch seroprevalence.
3. Material and methods
3.1. Study design and sample collection
The study design and the sample collection have been described in Chapter 3.
3.2. Collection of questionnaire data and statistical method
The same questionnaire data and statistical method as described in Chapter 5 are used.
4. Results
An overview of the collected farm data showed that some factors were not useful for the
analysis due to different reasons. When there were 5 farms or less applying a certain
factor, it was excluded. Examples for exclusion were: being a Specific Pathogen Free farm
(only one SPF-farm), the use of a new set of clothes when changing house (only 3 farms)
and the use of acidified feed in the nursery (only 3 farms). This resulted in exclusion of 19
factors. Moreover, variables with a high number of missing values (more than 15 missing)
were omitted. Most of these missing values were due to the interviewed person, who was
in these cases often the keeper and not the owner of the stables or the keeper/owner did
not record certain information (e.g. daily weight gain).
Some variables were pooled, e.g. the factor ‘cleaning-disinfection-empty’ (‘CDE’) consists
of (1) the cleaning and (2) the disinfection of the stable after each rearing round and (3)
the time period in which the stable remained empty after the pigs were slaughtered (at
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least 3 days). A ‘proper use of a disinfection bath’ exists of (1) where the bath was placed
and (2) over what time period the bath was renewed.
According to the reducing measurements mentioned above, only 36 explanatory factors
were retained for the analyses (Table XIV).
Only 6 factors remained significant after the univariate analyses (P < 0.05). During the
multivariate analysis, the factors ‘pets allowed in the stable’ (univariate analysis: P-value
= 0.019) and the ‘proper us of a disinfection bath’ (univariate analysis: P-value = 0.031)
were eliminated. The final logistic regression model yielded three risk factors, one
protective factor and one significant interaction (Table XVI).
The risk factors were ‘the use of a semi-slatted floor in the fattening pig unit’, ‘presence
of other pig farms in the area (closer than 500m)’ and ‘the number of piglet suppliers’. As
protective factor there was ‘snout contact possible between pigs from adhering pens’
that, in relation with the presence of pets in the stable, turned into a risk factor.
Table XVI. Final logistic regression model with a random effect for farm, of variables
significantly (P ≤ 0.1) associated with the presence of antibodies against human
pathogenic Yersinia spp. in Belgian pig batches at slaughter (n = 100 farms).
Factor Odds ratio P-value 95% CI
Semi slatted floor in fattening pig unit 3.78 0.022 [1.21; 11.82]
Presence of other pig farms in the area (closer than 500m)
2.32 0.076 [1.01; 5.31]
Number of piglet suppliers 1.43 0.003 [1.13; 1.82]
Snout contact possible between pigs from adhering pens
0.10 0.001 [0.03; 0.37]
Interaction: snout contact and pets 17.64 0.004 [2.56; 121.51]
There were 41 farrow-to-finish farms included in this study, that had a mean within-batch
seroprevalence of 60% (Fig. XVII). The fattening pig farms with just one piglet supplier
(n=34) had a slightly higher prevalence of 64% (P > 0.1). The prevalence increased for
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farms with 2 (n=8), 3 (n=5) or more (n=12) piglet suppliers to 68, 78% and 89% respectively
(P < 0.1).
The mean within-batch seroprevalence of the assessed binary risk and protective factors
are shown in Fig. XVIII. Nineteen farms had a semi slatted floor in the fattening stables,
which resulted in a mean within-batch seroprevalence of 74%, whereas the other 81 farms
with a fully slatted floor had a mean seroprevalence of 65% (P < 0.1). When there were
other pig farms in the surroundings (n=63), these farms had a higher seroprevalence
compared to farms without other pig farms in the area (n=37) (75 and 60% respectively)
(P < 0.1). Farms with the possibility of snout contact between pigs in adherent pens (n=67)
obtained a lower seroprevalence (61%) compared to stables with a closed pen separation
(n=33; 78%) (P < 0.1). Looking closer to the interaction of snout contact and pets in the
stable, it is noticed that out of the 67 farms with possible snout contact, 27 did not allow
pets in the stables (Fig. XIX). These farms had a mean within-batch seroprevalence of 51%
(P < 0.1). The other 40 farms had a prevalence of 75% which is similar to the farms with
no possibility of snout contact in the fattening pig stables. Sixteen of them had pets in the
0
10
20
30
40
50
60
70
80
90
100
0
(n=41)
1
(n=34)
2
(n=8)
3
(n=5)≥4
(n=12)
mea
n w
ithin
-bat
ch s
eropre
val
ence
(%)
number of piglet suppliers
Figure XVII. Distribution of the mean within-batch seroprevalence (%) over the
different number of piglet suppliers per farm
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stable and had a mean seroprevalence of 74%, while the 17 remaining farms with no snout
contact and no pets had a mean within-batch seroprevalence of 81%.
0
10
20
30
40
50
60
70
80
90
semi slatted floorpresence of other
pig farms in the
area
possible snout
contact
pets allowed in
the stable
mea
n w
ithin
-bat
ch s
eropre
val
ence
(%
)
yes
no
0
10
20
30
40
50
60
70
80
90
snout contact+
pet+
(n=27)
snout contact+
pet-
(n=40)
snout contact-
pet+
(n=16)
snout contact-
pet-
(n=17)
mea
n w
ithin
-bat
ch s
eropre
val
ence
(%
)
Figure XVIII. Comparison of the mean within-batch seroprevalence between the binary
outcome of the assessed risk and protective factors.
Figure XIX. Comparison of the mean within-batch seroprevalence between the
four categories of possible snoutcontact and pets present in the stable.
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5. Discussion
The aim of this study was to investigate multiple risk factors for the presence of antibodies
against YOP’s of Yersinia spp. in meat juice of pigs at slaughter, while controlling for
clustering by farm. This analysis was based on the infection status of the pigs, specifying
a positive animal having an activity value of 30 OD%. The number of pigs sampled per
batch was established very precise and accurate (number of pigs to be sampled per batch
was calculated based on an expected batch prevalence of 50%, a confidence level of 95%
and an accepted error of 10%). The risk factors based on the serological results show the
possible risks for infection during the whole rearing period, even when the infection is
already finished, which is in contrast to risk factors based on the microbiological
prevalence at time of slaughter.
The first two risk factors, ‘the use of a semi-slatted floor in the fattening pig unit’ and
‘presence of other pig farms in the area (closer than 500m)’ could be explained easily.
Using a semi-slatted floor allows accumulation of faeces on the pen floor. Since pig faeces
can contain human pathogenic Yersinia spp., the contact between these faeces and pigs
may lead to infection (Fukushima et al., 1983; Nielsen et al., 1996; Nesbakken, 2006). The
use of a fully slatted floor was mentioned as a protective factor by von Altrock et al.
(2011), which is similar to the present study. The presence of other pig farms closer than
500m to the investigated farm is also a risk factor for an increasing prevalence. This could
be due to close contact between farmers, the nearby passage of farm vehicles or even the
spread by rodents (Backhans et al., 2011). These two factors are difficult to adjust by the
farmer.
The third risk factor in the current study, ‘the number of piglet suppliers’, was also noted
by Vilar et al. (2013) and may also represent a difference between the farrow-to-finish
farms (zero suppliers) and the fattening pig farms (1-11 suppliers in the current study).
Figure XVII shows the more piglet suppliers there are at one farm, the higher the
seroprevalence is. Skjerve et al. (1998) indicated being a farrow-to-finish farm is a
protective factor, which also aligns to the present study. Virtanen et al. (2012) showed
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that incoming piglets, possibly infected on their multiplying farm of origin, are a feasible
source of infection for other piglets originating from other farms. The more piglets are
purchased by different suppliers, the greater the risk of buying infected pigs and of
spreading Yersinia spp. in the pen.
The only protective factor that was present in the current study was ‘snout contact
possible between pigs from adhering pens’. This contact could lead to a higher chance of
spread between pens, leading to a higher prevalence. Nevertheless, if this snout contact
could lead to infections at very young age, resulting in a decrease of antibody response
below the cut-off value by the time the pigs are slaughtered. A study of Nesbakken et al.
(2006) mentioned an increasing level of titers from an age of 80 days to 162 days in two
multiplying herds, and a study of Fukushima et al. (1983) presented an increasing level of
seropositive pigs at day 77. But when pigs are slaughtered at an age of 6-6.5 months (183-
195 days), these levels may be already decreasing. Nielsen et al. (1996) showed a
decreasing antibody titer over time. In the study of Virtanen et al. (2012), based on the
microbiological prevalence, snout contact was mentioned a risk factor. It is possible that
the tonsils remain infected, while the antibody titer is decreasing. If there is a pet allowed
in the stable, the possible snout contact becomes a risk factor. Pets can enter different
stables and may carry pathogens originating from these stables, possibly resulting in a
continuous re-infection (Yanagawa et al., 1977; Fredriksson-Ahomaa, 2001; Murphy et al.,
2010). Nevertheless, Fukushima et al. (1983) did not find an increasing antibody titer after
re-infection. More research is needed about the fluctuation of antibody titer troughout
the rearing period and after re-infection. Moreover, a comparison between
microbiological en serological results at pig- and batch-level should be made.
A limited number of studies about risk factors at farm-level were based on a serological
prevalence (Skjerve et al., 1998; von Altrock et al., 2011). In these studies, other risk
factors were presented. Straw bedding was categorized as a risk factor in these studies,
but such a bedding is not common in Belgian pig farms (only applied by one farm in this
research). The use of municipal water was regarded as a protective factor in these studies,
but was not significant in the present study.
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At pig farm level, there is a second foodborne zoonosis to be taken into account. The risk
factors of a high seroprevalence of Salmonella have been assessed many times. The
identified risk factors are: the moving of individual animals during the fattening period,
not having a separate transporter for different age groups, pigs having contact to other
animals, application of antibiotics, being situated less than 2 km away from another pig
farm, an increased number of pig farms within a 10 km radius and the use of granulated
feed instead of flour. The following factors were identified as being protective: not
cleaning the transporter, not having clean boots available, fully slatted floor, use of
protective clothing, cleaning the feed tube, administer liquid feeding instead of dry
feeding, feeding homemix and barley and being a conventional pig farm (Hotes et al.,
2010; Smith et al., 2010; Gotter et al., 2012). Some of these factors (the proximity of other
pig farms and the fully slatted floor) affect the antibody level of both Salmonella as
Yersinia spp. von Altrock et al. (2011) insinuated that the Yersinia seroprevalence is
inversely associated with the serological Salmonella status. This has to be taken into
account when setting up a combined zoonotic control program for Salmonella and
Yersinia.
To conclude, in the present study, farm factors influencing the seroprevalence of human
pathogenic Yersinia spp. in pig batches at slaughter, were analyzed. Determined risk
factors are the use of a semi slatted floor in the fattening pig unit, the presence of other
pig farms in the area, an increasing number of piglet suppliers and the possible snout
contact in the presence of pets (interaction). The only protective factor is the possibility
of snout contact in the fattening pig unit. When using this knowledge to change some
factors in pig farms, the prevalence at farm level should decrease.
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GENERAL DISCUSSION
GENERAL DISCUSSION ________________________________________________________________________
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128
Yersiniosis is in Europe and the U.S. still one of the most common bacterial zoonosis due
to consumption of contaminated food (Scallan et al., 2013; EFSA and ECDC, 2013), with
pork identified as the most important source for human infection (Tauxe et al., 1987;
Ostroff et al., 1994; Fredriksson-Ahomaa et al., 2006; Boqvist et al., 2009; Huovinen et al.,
2010). Therefore, the general aim of this thesis was to clarify the infection status of pigs
with enteropathogenic Yersinia spp. at slaughter age and to assess the factors influencing
the prevalence.
The (cross-)contamination of pork mostly occurs during transformation of the meat as the
bacteria can still be present in the throat and less frequent in the faeces of infected pigs
presented for slaughter (Laukkanen et al., 2009). A reduction of the number of human
cases could be accomplished when less pig carcasses would be contaminated. It has been
demonstrated that hygienic measurements in the slaughterhouse alone will fail to
significantly reduce the carcass contamination (Nesbakken et al., 1994; Laukkanen et al.,
2010b; Ranta et al., 2010). Therefore, lowering the number of infected pigs prior to
slaughter is a major intervention strategy. This requires however as a start basic
information about the prevalence at slaughter age as well as a predictive tool for this
prevalence. This tool can help to identify the infection status of pig batches at slaughter
prior to slaughter, which can influence the order of slaughtering.
1. A predictive tool for the infection status at slaughter age
To determine the prevalence of enteropathogenic Yersinia spp. in pig batches, two
methods are currently applicable. Microbiological analysis indicates a momentary
infection, while serology symbolizes previous infection(s). Microbiological analysis is
based on the detection of Yersinia in two different matrices: faeces or tonsils. Analyzing
faeces at slaughter age is possible prior to slaughter in contrast to analyzing tonsils.
Collecting a piece of the tonsils in the live animal may negatively affect animal welfare.
Faecal analysis is however less relevant than studying tonsils because the faecal shedding
has ended while the infected tonsils are still detectable (Nielsen et al., 1996; Nesbakken
GENERAL DISCUSSION ________________________________________________________________________
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129
et al., 2006; Virtanen et al., 2012). Therefore, studies in the thesis focused on the tonsils
as matrix to detect the presence of Yersinia spp.
Based on the microbiological analysis of tonsils, a large variation seems to be present in
the within-batch prevalence of pig batches at slaughter (Chapters 1 and 2). Reasons for
this variation are, however, still unknown. The moment of initial infection is likely to be of
major significance. In batches with low microbiologically based prevalence, it is not certain
if this is due to a recent infection with a low number of animals infected, or if the infection
started long time ago with still some infected animals present. The porcine tonsils could
also become contaminated during transport or in the lairage when pigs are picking up
contaminated faeces. When batches are delivered to the slaughterhouse, some infected
pigs will still shed the bacteria in the faeces. In this way, they can infect other pigs or
contaminate the pens of the lairage area, and infect pigs of subsequent batches that enter
these pens. Nevertheless, since the faeces contain a low number of Yersinia bacteria at
moment of slaughter (Nesbakken et al., 2003; Nesbakken et al., 2006; Van Damme, 2013),
the number of Yersinia found in the tonsils at the slaughterline after possible
contamination during transport or in the lairage will likely also be low. When observing
the Yersinia count of microbiologically positive and serologically negative pigs (Fig. XX),
121 out of the 159 were harboring a high Yersinia count (> 3 log10 CFU/g tonsil). The mean
Yersinia count in pig faeces is 3 log10 CFU/g faeces (Van Damme et al., 2013). The time of
transportation and the time spent in the lairage are too short for the Yersinia to reach
large numbers (growth rate: 33-39 min at 32°C) (Schiemann, 1980). This suggests that
these 121 microbiologically positive and serologically negative pigs were likely infected
(serologically negative), shortly before transport to slaughter (count was too high).
GENERAL DISCUSSION ________________________________________________________________________
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130
Fig. XX. The Yersinia count of microbiologically positive and serologically negative
(activity value < 30 OD%) pigs.
Reducing the risk of contamination in the lairage, cleaning and disinfecting (a part of) the
lairage are interesting options. However, from a practical viewpoint, this is impossible to
accomplish during the slaughter day. Consequently, the (small) risk to infect new pigs in
the lairage will remain. Nevertheless, when negative batches would be slaughtered first
(preferably at the beginning of the week), and there would be a thorough cleaning and
disinfection at the end of every day or at least every week, the probability that pigs would
get infected in the lairage should already decrease.
Also a wide range (0 to 64.4%) of the microbiological prevalence was observed without
clustering around certain percentages, which implies that there is no accurate distinction
possible between low and highly infected batches. Another possibility to make a
distinction based on infection rate is the use of serology. Therefore, two matrices are
available. Blood can be obtained prior to and during slaughter, in contrast to meat juice
that can only be collected after slaughter. According to Meemken and Blaha (2011), these
two matrices show an excellent agreement, hence meat juice can be used instead of
blood. In Chapter 3, the observed within-batch seroprevalence showed an even larger
variation (0 to 100%) than the microbiological prevalence. The comparison of the
microbiological and serological within-batch prevalence resulted in similarities as well as
-5
0
5
10
15
20
25
30
35
0 1 2 3 4 5 6 7
acti
vit
y v
alu
e (O
D%
)
Yersinia count (log10 CFU/g tonsil)
GENERAL DISCUSSION ________________________________________________________________________
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discrepancies (Chapter 4): there were batches microbiologically negative and serologically
negative (13%), which implies that these farms are enteropathogenic Yersinia spp. free. A
low seroprevalence always indicates a low microbiological prevalence though not
necessarily Yersinia free. Using serology to distinguish low and highly infected batches, is
possible. However, a low microbiological prevalence can imply a low or a high
seroprevalence. Batches with a low microbiological prevalence and low seroprevalence
point towards a recent infection, it is even possible this happened in the lairage (Nielssen
et al., 1996; Nesbakken et al., 2006), while a low microbiological prevalence combined
with a high seroprevalence suggests an infection that took place earlier. The analytical
results at slaughter age depend on the moment of the initial infection and on its course.
It is not well known yet at which specific age pigs become infected. If piglets introduce
Yersinia spp. in pig farms (Virtanen et al., 2014), the infection is expected to take place at
a young age. Moreover, according to Fukushima et al. (1983), who experimentally re-
infected pigs, Yersinia spp. did not reappear in the faeces, concluding that re-infection
does not occur. The influence of a second infection on the infection status of the tonsils
is still unknown. Consequently, without re-infection and after clearing the infection, many
pigs should become Yersinia free prior to slaughter. In the present studies however, many
pigs are still infected in their tonsils prior to slaughter. This could be due to re-infection or
to the carrier status of the pigs. In contrast to the hypothesis of Fukushima et al. (1983),
re-infection probably does occur. The study of Fukushima et al. (1983) was performed a
long time ago. It would be interesting to verify whether the results are still applicable and
to further elucidate the possibility of re-infection. Secondly, if re-infection exists, its
frequency will have an influence on the bacteriological and serological prevalence at the
moment of slaughter.
The implementation of the prediction of the infection rate of pigs prior to slaughter by
serology is matrix depending. The advantages of using serology are the low cost, and the
applicability prior to slaughter while microbiological analysis of the tonsils is more
expensive, time consuming and can only be carried out after slaughter. When the
information about the prevalence is available after slaughter, it is already too late to
differentiate between low and highly infected batches prior to slaughter. Also, it is too
GENERAL DISCUSSION ________________________________________________________________________
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132
late to avoid (cross-)contamination at the slaughterline and to have an impact on the meat
contamination status. A low seroprevalence was always related to a low microbiological
prevalence (Chapter 4). In contrast, a high seroprevalence stands for either a low or a high
microbiological prevalence. Before the collection of data overtime (historical data) can be
introduced, the fluctuation of the prevalence of subsequent batches from the same farms
should be studied. For instance, it could be that the batches coming from the same farm
will always show the similar prevalence. However, it is also possible that there is a big
variation between these batches. Using historical data based on microbiology of tonsils to
categorize the following batches is only useful when the within-batch microbiological
prevalence is stable over time. This should be studied. The result should be included in
the food chain information document. The farmer can assess the infection rate and the
effect of preventive measurements by checking the results of the within-batch prevalence
provided by the slaughterhouse.
Since batches with a low seroprevalence also have a low microbiological prevalence,
serological analysis is a tool to assure the microbiological prevalence at slaughter.
Serological based categorization of batches with a low or high infection rate, creates the
opportunity of logistic slaughtering, which is a new possibility to lower the risk of (cross-)
contamination in the slaughterhouse. The formulated equation in Chapter 4 is the basis
to this categorization.
𝑚𝑒𝑎𝑛 𝑤𝑖𝑡ℎ𝑖𝑛 − 𝑏𝑎𝑡𝑐ℎ 𝑚𝑖𝑐𝑟𝑜𝑏𝑖𝑜𝑙𝑜𝑔𝑖𝑐𝑎𝑙 𝑝𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 = 0.444
1 − 𝑒−0.063∗(𝑂𝐷%−37.069)
This equation seems difficult, but it is usable. When a farm for example has a mean activity
value of 20 OD%, the mean within-batch microbiological prevalence is -0.23, which means
there are no infected pigs in this batch. When, on the other hand, the mean activity value
is 50 OD%, the mean within-batch microbiological prevalence is 0.80. Many pigs in this
batch are infected at slaughter age.
GENERAL DISCUSSION ________________________________________________________________________
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133
2. The possibilities of the farmer to influence the prevalence
At farm level, potential opportunities are present to decrease the number of infected pigs
delivered to the slaughterhouse. Risk and protective factors were studied in Chapters 5
and 6. Three risk factors were the same in both studies: the use of a semi slatted floor,
the presence of pig farms in the area and the number of piglet suppliers. Two factors, the
proper use of a disinfection bath and the possible snout contact between pigs from
adherent pens, were significant protective factors in the multivariate analysis based on
the microbiological and serological prevalence, respectively, while they were both
significant protective factors in the univariate analysis of the other study. The CDE is only
a significant protective factor in the study based on the microbiological prevalence. CDE
and the possible snout contact both interact with the presence of pets, resulting in the
change from protective to risk factor. Differences between the results of the
microbiological and serological risk factor study have two explanations. Firstly, the species
on which the study was based on. The microbiological factors are based only on the
prevalence of Y. enterocolitica, while the serological factors are based on human
pathogenic Yersinia spp. Secondly, the microbiologically based risk factors indicate the
possibilities why pigs could be infected at moment of slaughter, while the factors based
on serology show the factors why pigs could get infected during rearing. Pig farmers
should take into account the risk and protective factors, which are indicated in this thesis,
to reduce the number of infected pigs. Some factors cannot be implemented straight
away. The proximity of pig farms in the area (closer than 500m) is not usable because the
location of a farm cannot be changed. This factor is a borderline risk factor (0.05 < P-value
< 0.1) in the study based on the serological prevalence, but is significantly associated
(Odds Ratio = 1.63; P-value = 0.036) in the risk factor study based on the microbiological
prevalence. Nevertheless, this factor was never identified as a risk factor in other studies,
maybe because some regions or countries have a very low density of pig farms (e.g.
Finland). There is still no clarification why this factor was significant in the present study.
The transmission of enteropathogenic Yersinia spp. between pig farms (e.g. emission of
air, pest animals, persons,…) has not been studied so far, except for transporting piglets
between farms (Virtanen et al., 2014). Yersinia spp. are maybe transferred aerogenic. In
GENERAL DISCUSSION ________________________________________________________________________
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the present study, only the proximity of the neighbours (500 m) was included. As
supposed by the fact that nearby farms are a risk factor for infection, there are possible
transmission routes. The direction of wind, the number of neighbours or their biosecurity
were not taken into account in this study. Theses three items are however important
when try to elucidate the influence of aerogenic spread. Yersinia spp. could also be
transferred by pest animals (insects or rodents), be due to contact between farms or a
minor biosecurity (e.g. same boots used in different compartments or contaminated
boots worn by visiting farmers). The possible transmission routes should be examined
more in detail. Pest animals could move between farms and transfer pathogens. Contact
between farms, especially between low and high infected farms, should be minimized. In
the studies described in Chapters 5 and 6, many biosecurity measurements showed to be
not significant. Even when biosecurity was at a high level, farms could still be Yersinia
positive. When a compartment contains only pigs from the same origin, and the internal
biosecurity is maintained, there should be no transmission possible between
compartments. Even these farms harbored pigs microbiologically positive in the tonsils.
Maybe these pigs were already infected when they arrived at the farm or were infected
by their mother. Another possibility is that Yersinia spp. could be transferred by air, by
flies or is part of the farm microbiota. The possibility of Yersinia staying on-farm for years
has never been investigated. The infection route at farms with a high biosecurity level is
still unknown. When the farmer does not use an all-in/all-out system, newly introduced
pigs can easily be infected by the elderly animals. However, this factor showed to be not
significant and many (80%) farmers did apply an all-in/all-out system in the fattening pig
unit. Pigs introduced in an empty compartment are still at risk for infection. Genotyping
Yersinia strains of subsequent batches of a fattening pig farm could indicate the possibility
of persisting strains. Also genotyping strains of nearby farms could demonstrate possible
transmissions between these farms.
Changing a semi into a fully slatted floor type and creating the possibility of snout contact
between pens in the fattening pig unit can only be implemented when the farmer is willing
to cooperate or when building or renewing a stable. This could however be introduced in
the legislation when building new stables. The use of a fully slatted floor was also
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indicated as a risk factor for a higher prevalence of Salmonella by Hotes et al. (2010). The
use of a semi slatted floor was a borderline risk factor (0.05 < P-value < 0.1) in the study
based on the microbiological prevalence of tonsils. But this same factor was strongly
associated (Odds Ratio = 3.78) as risk factor to the serological prevalence. This suggests
that this factor is meaningful in both studies. Laukkanen et al. (2009) indicated the use of
bedding material as a protective factor. Nevertheless, the use of bedding material is not
compatible with a fully slatted floor. The possible snout contact in the fattening pig unit
is strongly associated with a lower seroprevalence (Odds Ratio = 0.1), while this factor was
only significant in the univariate analysis based on microbiological prevalence (Odds Ratio
= 0.6). The low seroprevalence of pigs raised on farms allowing snout contact, points to
an infection at a young age (antibodies are decreasing) or very recently (still low antibody
levels). A very recent infection due to snout contact is unlikely since the pigs are having
this contact since they entered the fattening pig unit. In the univariate analysis based on
microbiological prevalence it seems that snout contact is also a protective factor. In the
multivariate analysis, the factor was not significant anymore. Distinguishing low from
highly infected farms at moment of slaughter based on serology is difficult in farms
allowing snout contact. They have more chance of having a low seroprevalence, but there
is no relation with the microbiological prevalence.
An applicable option at every pig farm to reduce the number of on-farm infections with
enteropathogenic Yersinia spp., is placing a disinfection bath inside, at the entrance of the
stable and replace the fluid at least every two weeks, based on the time of activity of the
products used (e.g. MS Kiemkill tabs, Schippers, Arendonk, Belgium). The use of a
disinfection bath was a significant protective factor (Odds Ratio = 0.58) in the risk factor
analysis based on the microbiological prevalence and also a significant protective factor
in the univariate analysis based on the seroprevalence (Odds Ratio = 0.31) (Chapters 5 and
6). Both studies indicate that the proper use of a disinfection bath will decrease the
prevalence. This is a low-cost effective measurement that also helps to avoid or diminish
the spread of other pathogens.
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An increasing number of piglet suppliers was indicated in both studies as a risk factor
(Odds Ratio = 1.15 in Chapter 5; Odds Ratio = 1.43 in Chapter 6). Decreasing the number
of piglet suppliers is an easily applicable measure to reduce the microbiologically and
serologically based prevalence and was also mentioned by Vilar et al. (2013). The
difference of the within-batch prevalence of farms with one versus two or three suppliers
indicates that though even with a minor effort, it is effective in reducing this prevalence.
This is probably due to the smaller chance of buying piglets from an infected farm. When
reducing the number of suppliers, it is also important that farmers select the Yersinia
negative suppliers. If piglets from different suppliers should be placed in different
compartments, with a satisfying internal biosecurity, the risk of spread will decrease.
In the thesis, there were some contradictions with the literature, as some factors studied
were not significant to have an influence on the microbiologically or serologically based
within-batch prevalence, whereas they were significant in other studies. The presence of
pest animals in the stables, for example, was not significant in the present study.
However, Laukkanen et al. (2009) found that their presence was a protective factor, while
Novoslavskij et al. (2013) indicated it as a risk factor. Another factor, like the feed
producer, turned out to be too diverse to take into account. Finally, Virtanen et al. (2011)
pointed fasting pigs before transport to the slaughterhouse as a risk factor for faecal
shedding at time of slaughter. Maybe, it is due to the stress induced by fasting, that starts
24 h before the transport to the slaughterhouse, that increases the faecal shedding of Y.
enterocolitica. However, farmers are obligated to fasten all pigs at slaughter before
transport.
Yersinia enterocolitica free pig herds can however be raised. Such herds have already been
established and maintained for many years in Norway, where Specific Pathogen Free (SPF)
herds do not contain Y. enterocolitica (Nesbakken et al., 2007). When the top of a
breeding pyramid is free from human pathogenic Y. enterocolitica, the prevalence of Y.
enterocolitica will decrease in the general pig population. The proportion of infected sows
variates between 0-14% (Niskanen et al., 2002; Korte et al., 2004; Gürtler et al., 2005;
Bowman et al., 2007; Wehebrink et al., 2008; Farzan et al., 2010). Keeping batches or
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herds non- or low infected, is also depending on the risk of transmitting Y. enterocolitica
to this farm. Pilon et al. (2000) has found a limited number of positive environmental
samples, indicating that infections are potentially coming from outside (pigs, persons or
pets). Nesbakken et al. (2007) suggested that these human pathogenic Y. enterocolitica–
free segments of the pig population could be the beginning of providing human
pathogen–free (HPF) pork on the market. At this time, the Belgian Federal Agency for the
Safety of the Food Chain (FAVV) is trying to produce Salmonella-free piglets by clearing
the top of the breeding pyramid from Salmonella (Sci Com 2013/06). As stated by
Nesbakken et al. (2007), pig herds can also become Y. enterocolitica-free. Maybe the
methods used to reduce the prevalence of Salmonella, may be similar to those decreasing
the prevalence of Yersinia spp.. Producing slaughter pigs free from both important
pigborne pathogens should be possible. It is still uncertain whether the goal of producing
Salmonella- and Y. enterocolitica-free slaughter pigs could be achieved by only reducing
the prevalence of both pathogens in the breeding herds and reduce or eliminate the
transfer of these pathogens in the feed. Other transmission routes could also be
important.
3. Final impact on carcass contamination
The slaughterhouse can adopt its slaughter activities on the within-batch prevalence of
arriving pigs at slaughter and apply appropriate slaughter techniques and logistic
slaughtering.
As mentioned by EFSA, and if pig batches are categorized according to the risk for human
health they imply, there are two options. First, the low infected pig batches could be
slaughtered, followed by the high infected batches. Then, it is of great importance to
obtain a strict separation between these two types of batches and the lairage area should
be cleaned thoroughly every day. Another possibility is to slaughter them on different
days. Logistic slaughtering has already been introduced for high Salmonella prevalence in
pig herds. This logistic slaughtering is only based on the seroprevalence of Salmonella,
without a relation with the microbiological prevalence. This resulted in the observation of
no clear effects on carcass contamination (Arguello et al., 2013). The seroprevalence does
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not indicate the infection rate of a pig batch at slaughter. Slaughtering batches having a
low seroprevalence still ends by slaughtering low and highly infected batches, which
results in an equal risk to contaminate carcasses during slaughter procedures as when all
batches are slaughtered independent of their serological status.
Adapting slaughtering techniques can be advantageous to avoid (cross-)contamination to
pig carcasses. Nesbakken et al. (1984) and Laukkanen et al. (2010) showed that bagging
of the rectum assists in the reduction of the faecal contamination of the carcasses. This
method is common in the Nordic countries. This practice is also useful to protect the
carcass from contamination with other bacteria. The tonsils are also an important source
of carcass contamination and the splitting machine is a transporter of bacteria from one
pig to another when the head is also splitted (Van Damme, 2013). In order to avoid carcass
contamination by the tonsils, EFSA suggests to open the head away from the slaughter
line. This practice however, has never been investigated thoroughly. Nesbakken et al.
(2003) showed that the incision of the submaxillary lymph nodes and touching carcasses
by the meat inspection personnel also allowed (cross-)contamination. At last, the previous
meat inspection was adapted to avoid transferring as less as possible bacteria between
pig carcasses by preventing contact of the inspector with the carcass. According to EFSA
(Biohaz, 2011), palpating and making incisions in the carcass is a risk factor of (cross-)
contamination. At this moment, only a visual meat inspection is allowed (EC 219/2014).
Carcasses are only palpated and incised when abnormalities have been discovered and
when the pig is removed from the slaughter line.
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4. Future perspectives
There are still some uncertainties remaining on the infection of pigs with
enteropathogenic Yersinia spp. which can be studied in the future:
- When does the pig get infected for the first time? Where does this initial infection
come from? Is the moment farm-dependent? Is there a difference between
farrow-to-finish and fattening pig farms?
- Do pigs get re-infected? If it exists, where does this infection comes from? How
does the pig react on this second infection?
- Concerning both mentioned future studies, the possible transmission routes of
infection are still unknown.
- What is the antimicrobial resistance of Y. enterocolitica? Is there a difference
between farms? Is this linked to the antimicrobial use on a farm?
- Is the introduction of Y. enterocolitica on a farm accompanied by Salmonella?
- Nearby farms are indicated as a risk factor. It is possible that those farms harbor
the same genotypes of Yersinia spp. This can also indicate a possible transmission
route.
- Does the prevalence fluctuate between subsequent batches? Maybe historical
data can be used to categorize batches.
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SUMMARY
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There are three human pathogenic species in the genus Yersinia: Y. pestis, Y.
pseudotuberculosis and Y. enterocolitica. While Y. pestis is no longer present in Europe,
human pathogenic Y. enterocolitica is the most important of the two enteropathogenic
Yersinia spp. due to its high number of human cases. The animal host species of Y.
pseudotuberculosis are birds and pest animals, whereas Y. enterocolitica is mostly found
in pigs. This latter species is also responsible for most of the foodborne outbreaks caused
by Yersinia spp. , originating from pork products whereas the outbreaks caused by Y.
pseudotuberculosis are mostly derived from vegetables. The prevalence of human
pathogenic Y. enterocolitica in pigs varies greatly among countries and pig farms (0-100%),
but statistically based studies about the within-batch prevalence were lacking. The way of
farming could have an influence on the presence of Y. enterocolitica.
A preliminary study of the within-batch prevalence of Y. enterocolitica in Belgian pig
batches at slaughter was performed to show the variation of this prevalence and is
discussed in Chapter 1. Tonsils of 1397 pigs from 66 batches were collected. On average
21 tonsils per batch were sampled. These tonsils were analyzed using the direct plating
method on cefsulodin-irgasan-novobiocin (CIN) agar plates, and these results were
verified performing a Polymerase Chain Reaction (PCR) (detecting the ail gene).
Pathogenic Y. enterocolitica were found in 375 pig tonsils (26.8%). The within-batch
prevalence ranged from 0 (20 batches) to 83.3%. The number of colonies varied between
2.01 and 6.00 log10 CFU g-1 tonsil. This preliminary study revealed a great variety in
presence of Y. enterocolitica among pig batches without clustering around a certain
prevalence.
These results were taken into account in the following study, where more samples per
batch were taken to detect low infected farms. The number of pigs per batch sampled
was calculated based on an expected batch prevalence of 50%, a confidence level of 95%
and an accepted error of 10%. Tonsils (Chapter 2) and pieces of diaphragm (Chapter 3)
were collected from each of the 7047 fattening pigs at slaughter, originating from 100
farms. On average, 70 pigs were sampled per batch. Again, direct plating on CIN agar
plates was performed on the homogenate of the tonsils, followed by a PCR(species and
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serotype). The pieces of the diaphragm (± 10g) were first stored at -20°C, where after they
were thawed and 2 ml of meat juice was collected, which was used in the ELISA Pigtype
Yopscreen (Labor Diagnostik Leipzig, Qiagen, Leipzig, Germany). This ELISA targets
antibodies against all human pathogenic Yersinia spp. The optical density (OD) was
determined at 450 nm. The results were positive if the absorbance exceeded the
proposed cut-off value of 30%. Pathogenic Y. enterocolitica serotype O:3 were found in
tonsils of 2009 pigs (28.5%), originating from 85 different farms, with a count between
2.01 and 5.98 log10 CFU g-1 tonsil (on average 4.00 log10 CFU g-1 tonsil). The within-batch
prevalence in positive farms ranged from 5.1 to 64.4%. Yersinia pseudotuberculosis was
found in seven farms, for which the within-batch prevalence varied from 2 to 10%. The
serological tests resulted in a binomial-shaped distribution with modes at 0-8.3 OD% and
58.3-66.6 OD% for the individual pigs with an average of 51 OD%. Sixty-six percent of the
animals tested positive according to the used criterion. At batch level, there was also a
binomial distribution with modes at 0-5% (n=12) and 85-90% (n=16). The within-batch
seroprevalence ranged from 0 (n=7) to 100% (n=1). The results of these studies revealed
that many batches are microbiologically as well as serologically positive for Y.
enterocolitica and Y. pseudotuberculosis and that there is a difference between farms.
This variation could be due to certain farm factors.
The two methods, microbiology and serology, of obtaining the within-batch prevalence
were compared (Chapter 4). If there is a relation, the microbiological prevalence at time
of slaughter and important for food safety, can be predicted by the serological prevalence
obtained before slaughter. The results of these prevalences were compared using a
mixed-effect logistic regression at pig and batch level. Of the 2009 pigs positive for Y.
enterocolitica, 1872 also had antibodies against Yersinia spp. Unfortunately, at pig level,
the microbiological contamination could not be predicted by the presence of antibodies.
Nevertheless, at batch level, a relation was observed (prevalence =0.444/(1-e-0.063*(Optical
Density-37.069)), cut-off value for a positive farm is 37 OD%). This formula could predict
whether a pig batch will contain infected pigs before they arrive at the slaughterhouse.
This way eventually, infected batches could be slaughtered last so the risk of cross-
contamination in the slaughterhouse could be avoided or diminished.
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The results of the study on the within-batch prevalence of Y. enterocolitica (and Y.
pseudotuberculosis) in pigs at slaughter were the basis of the risk factor analysis at farm
level. In Chapter 5, the analysis was performed on the bacteriological results discussed in
Chapter 2, but only based on Y. enterocolitica. Chapter 6 is looking at the risk factors for
a high seroprevalence, based on both enteropathogenic Yersinia spp. Each farm was
visited and data concerning housing, ventilation, biosecurity, management, feeding and
disease control were collected using a face-to-face questionnaire. The number of positive
animals per batch was the outcome variable. First, variables were submitted to a
univariable analysis using a mixed effect logistic regression, with farm as random effect.
Variables which were related to the Yersinia prevalence (P < 0.05) were included in a
multivariable model, excluding at each step the non-significant variable until only
significant main effects and interactions remained. In the multivariable model based on
bacteriology, three risk factors, two protective factors and one interaction remained
significantly associated with Y. enterocolitica carriage in the tonsils (P < 0.1). More piglet
suppliers, a high density of pig farms in the surroundings and semi slatted floors in the
fattening pig stables were positively associated with a higher infection level whereas the
use of a disinfection bath before entering the stables and a poor biosecurity level were
protective factors. The latter protective factor showed a significant interaction with the
factor ‘presence of pets in the stables’, which increases the prevalence. Based on
serological results, four risk factors, one protective factor and one interaction remained
significantly associated with the presence of antibodies against enteropathogenic Yersinia
spp. in meat juice (P < 0.1). Many piglet suppliers, a high density of pig farms in the area
and the use of semi slatted floors in the fattening pig unit were considered risk factors.
The only protective factor was the possibility of snout contact in the fattening pig unit,
although a significant positive interaction between the presence of pets in the stables and
snout contact was observed. The risk factors are similar between microbiological and
serological prevalence results. Nevertheless, a poor biosecurity level has an influence on
the bacteriological prevalence, but not on the serological prevalence. Otherwise, snout
contact decreases the antibody level in pig batches, but has no influence on the presence
of Y. enterocolitica in tonsils of pigs at slaughter.
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At last, a proposal is formulated in the general discussion to reduce the amount of
contaminated carcasses in the slaughterhouse. This proposal is based on a control at farm-
and slaughterhouse level. The pig farmer can, after analyzing the risk and protective
factors, indicate what has to/should be changed at the farm. At the end of the rearing
period, blood samples should be collected to obtain an impression of the infection rate in
a batch of slaughter pigs. The results can help the slaughterhouse to indicate non- or low-
infected batches and to slaughter them first, so contamination in the slaughterhouse can
be reduced. More research should be performed about the dynamics of infection at farm
level, what happens with the antibody titer after re-infection and subsequent batches of
the same farm should be observed to indicate the variation of the prevalence over time.
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SAMENVATTING
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Binnen het genus Yersinia bestaan er 15 species die geen infectie bij de mens kunnen
veroorzaken en drie humaan pathogene species: Y. pestis, Y. pseudotuberculosis en Y.
enterocolitica. Alle drie bezitten ze minstens één virulentieplasmide, dat naast de
chromosomale virulentiegenen, verantwoordelijk zijn voor het ontstaan van infecties.
Yersinia enterocolitica is onderverdeeld in zes biotypes, waarvan enkel 1A niet humaan
pathogeen is (mist het virulentieplasmide). Yersinia pestis is niet meer aanwezig in Europa
waardoor deze niet meer verder besproken wordt. De humaan pathogene Y.
enterocolitica is de belangrijkste van de twee enteropathogene Yersinia species door zijn
relatief hoog aantal humane casussen. Beiden geven gastro-intestinale symptomen. De
dierlijke gastheren van Y. pseudotuberculosis zijn vogels en knaagdieren, terwijl Y.
enterocolitica vooral teruggevonden wordt bij varkens. Dit laatste species is ook
verantwoordelijk voor de meeste voedselgebonden uitbraken veroorzaakt door Yersinia,
uitgaande van gecontamineerd varkensvlees. Uitbraken veroorzaakt door Y.
pseudotuberculosis zijn meestal het gevolg van de consumptie van besmette groenten.
De prevalentie van humaan pathogene Y. enterocolitica in varkens varieert sterk tussen
verschillende landen en varkensbedrijven (0-100%). Studies over de binnenlotprevalentie
gebaseerd op statistische aantallen waren er echter niet. De manier waarop een
varkensbedrijf gerund wordt, kan, naast de infrastructuur, een grote invloed hebben op
de aanwezigheid van Y. enterocolitica.
In een preliminaire studie bij verschillende loten van Belgische varkens werd aangetoond
dat er een variatie is in deze prevalentie op het moment van slachten (Hoofdstuk 1).
Tonsillen van 1397 varkens, afkomstig van 66 loten, werden verzameld. Gemiddeld
werden er 21 tonsillen per lot bemonsterd. Deze tonsillen werden geanalyseerd via de
directe uitplatingsmethode op cefsulodin-irgasan-novobiocin (CIN)-agar platen. Humaan
pathogene Y. enterocolitica werden teruggevonden in 375 varkenstonsillen (26,8%). De
binnenlotprevalentie varieerde van 0 (20 loten) tot 83,3%. Het aantal kolonies per gram
tonsillair weefsel varieerde van 2,01 tot 6,00 log10 CFU. Deze preliminaire studie toonde
een grote spreiding aan in de aanwezigheid van Y. enterocolitica tussen varkensloten
afkomstig van verschillende bedrijven waarbij ook een grote variatie aanwezig was (geen
clustering rond bepaalde prevalenties).
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Deze resultaten werden meegenomen naar een tweede studie, waar meer stalen per lot
werden genomen om laag-geïnfecteerde bedrijven te detecteren. Het te bemonsteren
aantal varkens per lot werd berekend op een verwachte binnenlotprevalentie van 50%
met een betrouwbaarheidsinterval van 95% en een aanvaarde fout van 10%. Tonsillen
(Hoofdstuk 2) en een stuk van het diafragma (Hoofdstuk 3) werden verzameld van elk van
de 7047 vleesvarkens op het moment van slachten. Deze varkens waren afkomstig van
100 loten die elk van een ander varkensbedrijf kwamen. Gemiddeld werden 70 varkens
per lot bemonsterd. Opnieuw werd het homogenaat van de tonsillen gebruikt bij de
directe uitplating op CIN-agar platen, gevolgd door een PCR op species- en
serotypeniveau. De stukjes diafragma (± 10g) werden eerst bewaard bij -20°C, waarna ze
ontdooid werden. Op het verzamelde vleesvocht (2 ml) werd de ELISA Pigtype Yopscreen
(Labor Diagnostik Leipzig, Qiagen, Leipzig, Germany) uitgevoerd. Deze ELISA detecteert
antistoffen tegen alle humaanpathogene Yersinia soorten. Wanneer de absorptie de
voorgesteld cut-off van 30% overschreed, waren de resultaten positief. Pathogene Y.
enterocolitica serotype O:3 werden teruggevonden in de tonsillen van 2009 varkens
(28.5%), komende van 85 verschillende bedrijven, met een telling tussen 2,01 en 5,98
log10 CFU g-1 tonsil (gemiddeld 4,00 log10 CFU g-1 tonsil). De binnenlotprevalentie in
positieve bedrijven varieerde van 5,1 tot 64,4%. Yersinia pseudotuberculosis werd
gevonden in zeven bedrijven. Daarbij varieerde de binnenlotprevalentie van 2 tot 10%. De
serologische tests resulteerden in een binomiaal gevormde distributie met modi bij 0-8,3
OD% en 58,3-66,6 OD% voor de individuele varkens, met een gemiddelde van 51 OD%.
Zesenzestig procent van de onderzochte dieren testte positief volgens het vooropgestelde
criterium. Op lotniveau was er eveneens een binomiale distributie met modi bij 0-5%
(n=12) en 85-90% (n=16). De binnenlotseroprevalentie varieerde van 0 (n=7) tot 100%
(n=1). Het resultaat van deze studies was dat veel bedrijven zowel microbiologisch als
serologisch positief zijn voor Y. enterocolitica en Y. pseudotuberculosis en dat er een
verschil is tussen bedrijven. De variatie zou het gevolg kunnen zijn van bedrijfsfactoren.
De beide methoden waarmee de prevalentie bepaald werd, werden vergeleken
(Hoofdstuk 4). Indien er een relatie is, kan de microbiologische prevalentie op het
moment van slachten, die belangrijk is voor de voedselveiligheid, voorspeld worden door
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de serologische prevalentie voor het slachten. De resultaten van deze prevalenties
werden vergeleken via een mixed-effect logistische regressie op dier- en lotniveau. Van
de 2009 varkens die positief waren voor Y. enterocolitica, hadden 1872 ook antistoffen
tegen Yersinia spp. Op dierniveau kon de microbiologische contaminatie niet voorspeld
worden aan de hand van de aanwezigheid van antistoffen. Daarentegen werd er op
lotniveau wel een relatie waargenomen (prevalentie =0.444/(1-e-0.063*(Optische Densiteit-
37.069))), met een cut-off waarde voor een positief bedrijf 37 OD%. Deze formule kan
voorspellen of een lot geïnfecteerde varkens zal bevatten voordat ze aankomen in het
slachthuis. Op deze manier kunnen de geïnfecteerde loten eventueel als laatste geslacht
worden zodat kruiscontaminatie in het slachthuis vermeden of verminderd kan worden.
De resultaten van de studie over de microbiologische en serologische
binnenlotprevalentie van Y. enterocolitica (en Y. pseudotuberculosis) in varkens op
slachtleeftijd vormden de basis van de risicofactoren analyse op bedrijfsniveau. In
Hoofdstuk 5 werd de analyse uitgevoerd op de bacteriologische resultaten van Y.
enterocolitica. Hoofdstuk 6 beschrijft de risicofactoren voor een hoge seroprevalentie,
gebaseerd op beide enteropathogene Yersinia spp. Elk varkensbedrijf werd persoonlijk
bezocht en info met betrekking tot het huisvesting, de ventilatie, de bioveiligheid, het
management, de voeding en de ziektebestrijding werden verzameld via een enquête met
de varkenshouder. Het aantal positieve dieren per lot was de uitkomst variabele. Eerst
werden de variabelen onderworpen aan een univariabele analyse gebruik makend van
een mixed-effect logistische regressie, met het bedrijf als random effect. Variabelen die
gerelateerd waren met de Yersinia prevalentie (P < 0.05) werden meegenomen in het
multivariabele model. Bij elke stap werden de niet-significante variabelen uitgesloten,
totdat enkel significante variabelen en interacties overbleven. In het multivariabele model
gebaseerd op bacteriologie, bleven drie risicofactoren, twee beschermende factoren en
één interactie over die significant geassocieerd waren met de aanwezigheid van Y.
enterocolitica in de tonsillen (P < 0.1). Meerdere biggenleveranciers, veel
varkensbedrijven in de omgeving (dichter dan 500m) en halfvolle vloeren in de
mestvarkensstallen waren positief geassocieerd met een hoger infectieniveau terwijl het
gebruik van een desinfectiebad voor het betreden van de stallen en een slechte
SAMENVATTING ________________________________________________________________________
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bioveiligheid beschermende factoren waren. Deze laatste vertoonde een positieve,
significante interactie met de factor ‘aanwezigheid van huisdieren in de stallen’.
Gebaseerd op de serologische resultaten, vier risicofactoren, één beschermende factor
en één interactie waren geassocieerd met de aanwezigheid van antistoffen tegen
enteropathogene Yersinia in vleessap (P < 0.1). Meerdere biggenleveranciers, veel
varkensbedrijven in de omgeving (dichter dan 500m) en halfvolle vloeren in de
mestvarkensstallen werden geïdentificeerd als risicofactoren. De enige beschermende
factor was de mogelijkheid van snuitcontact in de vleesvarkensstallen. Een significant
positieve interactie tussen de aanwezigheid van huisdieren in de stallen en snuitcontact
werd gezien. Deze risicofactoren zijn gelijklopend tussen de microbiologische en
serologische prevalentieresultaten. Hoe dan ook, een slechte bioveiligheid heeft een
invloed op de bacteriologische prevalentie, maar niet op de serologische resultaten. Aan
de andere kant, vermindert snuitcontact het niveau van aanwezige antistoffen in loten,
maar heeft geen invloed op de aanwezigheid van Y. enterocolitica in tonsillen van varkens
op slachtleeftijd.
Tot slot wordt er in de algemene discussie een voorstel gedaan dat kan leiden tot een
reductie van het aantal gecontamineerde karkassen in het slachthuis. Dit voorstel is
gebaseerd op een controle op bedrijfs- en slachthuisniveau. De varkenshouder kan, na
het analyseren van de risico- en beschermende factoren, bekijken wat er op zijn bedrijf
veranderd kan/moet worden. Op het einde van de vetmestingfase kan er per bedrijf bloed
verzameld worden om zo een indruk te krijgen van de infectiestatus van een lot varkens.
Deze uitslag kan gebruikt worden door het slachthuis om niet- of laag-besmette loten
eerder te slachten zodat contaminatie in het slachthuis gereduceerd kan worden.
Daarnaast moet er nog verder onderzoek gebeuren naar de dynamiek van de infectie
binnen een bedrijf, wat er met de antistoffentiter gebeurt na herinfectie en
opeenvolgende loten zouden opgevolgd moeten worden om eventuele variatie op te
sporen tussen loten van hetzelfde bedrijf.
________________________________________________________________________
153
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CURRICULUM VITAE
CURRICULUM VITAE ________________________________________________________________________
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186
Gerty Vanantwerpen werd geboren op 17 juni 1987 te Kortrijk. Eens afgestudeerd aan het
Spes Nostra Instituut te Heule, richting Latijn-Wiskunde, startte ze met haar studies
Diergeneeskunde aan de Universiteit Gent.
In 2011 behaalde zij met onderscheiding het diploma van Master in de Diergeneeskunde
(optie varken, pluimvee en konijn) en het FELASA certificaat type C in de proefdierkunde.
Haar Masterproef werd uitgevoerd in het Labo Hygiëne en Technologie van de Vakgroep
Veterinaire Volksgezondheid en Voedselveiligheid. Aangetrokken tot wetenschappelijk
onderzoek startte zij in datzelfde jaar een doctoraatsonderzoek. Op 1 november 2012
kreeg zij een assistentenplaats toegekend waardoor zij praktische oefeningen voor eerste
en derde master diergeneeskunde kon organiseren. In 2012 behaalde zij het diploma van
de opleiding ‘Food Packaging’ aan de UGent. In 2013 won zij de posterprijs op het
internationaal Yersinia congres in China. Tot slot vervolledigde zij de Doctoral Schools of
Life Sciences and Medicine van de UGent in 2014.
Gerty Vanantwerpen is auteur en mede-auteur van meerdere wetenschappelijke
publicaties in internationale tijdschriften. Ze nam actief deel aan meerdere internationale
en nationale congressen.
PUBLICATIES IN INTERNATIONALE TIJDSCHRIFTEN MET PEER-REVIEW
Vanantwerpen, G., Houf, K., Van Damme, I., Berkvens, D., De Zutter, L., 2013. Estimation
of the within-batch prevalence and quantification of human pathogenic Yersinia
enterocolitica in pigs at slaughter. Food Control 34, 9-12.
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf, K., 2014. Within-batch prevalence
and quantification of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis
in tonsils of pigs at slaughter. Veterinary Microbiology 169, 223-227.
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf, K., 2014. Seroprevalence of
enteropathogenic Yersinia spp. in pig batches at slaughter. Preventive Veterinary
Medicine.
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187
Van Damme, I., Vanantwerpen, G., Berkvens, D., De Zutter, L., 2014. Relation Between
Serology of Meat Juice and Bacteriology of Tonsils and Feces for the Detection of
Enteropathogenic Yersinia spp. in Pigs at Slaughter. Foodborne Pathogens and Disease 11,
596-601.
Vanantwerpen, G., Van Damme, I., Berkvens, D., De Zutter, L., Houf, K. Assessment of Risk
Factors for a High Within-batch Prevalence of Yersinia enterocolitica in Pigs based on
Microbiological Analysis at Slaughter. Under review at Veterinary Microbiology.
Vanantwerpen, G., Van Damme, I., Berkvens, D., De Zutter, L., Houf, K. Factors influencing
the risk of infection with human pathogenic Yersinia spp. during rearing of fattening pigs.
Under review at Epidemiology and Infection.
Vanantwerpen, G., Berkvens, D., De Zutter, L., Houf, K. Prediction of the infection status
of pigs and pig batches at slaughter with human pathogenic Yersinia spp. based on
serological data. In preparation.
Vanantwerpen, G., Houf, K., Berkvens, D., De Zutter, L. Assessment of the sampling
methodology on the detection of Salmonella contaminated pork carcasses. In
preparation.
Vanantwerpen, G., Vanthemsche, P., Landuyt, C., Barbier, J., De Zutter, L., Houf, K. Loss
of consciousness of cattle slaughtered by throat incision without stunning in a 180°
rotated position. In preparation.
Van Damme, I., Berkvens, D., Vanantwerpen, G., Wauters, G., De Zutter, L. Risk factors
associated with pig carcass contamination by enteropathogenic Yersinia spp. in Belgium.
In preparation.
CURRICULUM VITAE ________________________________________________________________________
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188
Vanantwerpen, G., Berkvens, D., De Zutter, L., Houf, K. Inter- and Intrafarm Comparison
of the Antimicrobial Resistance of Yersinia enterocolitica Isolated from Pigs. In
preparation.
ORALE PRESENTATIES
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf K. Risk factors for a high batch-level
bacteriologic prevalence of Y. enterocolitica in Belgian pigs at slaughter. ECVPH AGM and
Conference.19-20 September 2013, Turin, Italy.
Vanantwerpen, G., De Zutter, L. Listeria monocytogenes: betekenis voor de
volksgezondheid en voorkomen in voedselproductieketen. Studienamiddag: Listeria in de
voeding. 15 mei 2014, Instituut voor Permanente Vorming, Faculteit Diergeneeskunde,
Universiteit Gent.
Vanantwerpen, G., Berkvens, D., De Zutter, L., Houf, K. Association between
microbiological and serological prevalence of human pathogenic Yersinia enterocolitica in
pigs and pig batches. ECVPH AGM and Conference. 6-8 October 2014, Copenhagen,
Denmark.
PROCEEDINGS EN ABSTRACTS
2012:
Vanantwerpen, G., De Zutter, L. Estimation of the Within-batch Prevalence of Human
Pathogenic Yersinia Enterocolitica in Pigs at Slaughter. FoodMicro 2012. 3-6 September
2012, Istanbul, Turkey.
Vanantwerpen, G., De Zutter, L. Estimation of the Within-batch Prevalence of Human
Pathogenic Yersinia Enterocolitica in Pigs at Slaughter. Seventeenth Conference on Food
CURRICULUM VITAE ________________________________________________________________________
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189
Microbiology. Belgian Society for Food Microbiology. 20 -21 September 2012, Brussels,
Belgium.
Van Damme, I., Berkvens, D., Vanantwerpen, G., Baré, J., De Zutter, L. Risk factors for
carcass contamination with pathogenic Yersinia enterocolitica in Belgian pig
slaughterhouses. Seventeenth Conference on Food Microbiology. Belgian Society for Food
Microbiology. 20 -21 September 2012, Brussels, Belgium.
Vanantwerpen, G., De Zutter, L. Estimation of the Within-batch Prevalence of Human
Pathogenic Yersinia Enterocolitica in Pigs at Slaughter. International Pig Veterinary
Society-belgian branch. Study afternoon. November 23th 2012, Merelbeke, Belgium.
2013:
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Seroprevalence of
Enteropathogenic Yersinia spp. in Batches of Pigs at Slaughter. IAFP European Symposium
on Food Safety. 15-17 May 2013, Marseille, France.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Within-batch Prevalence and
Quantification of Human Enteropathogenic Yersinia spp. in Pigs at Slaughter. IAFP
European Symposium on Food Safety. 15-17 May 2013, Marseille, France.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Seroprevalence of
Enteropathogenic Yersinia spp. in Batches of Pigs at Slaughter. Yersinia 11. 24-28 June
2013, Suzhou, China.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Within-batch Prevalence and
Quantification of Human Enteropathogenic Yersinia spp. in Pigs at Slaughter. Yersinia 11.
24-28 June 2013, Suzhou, China.
CURRICULUM VITAE ________________________________________________________________________
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190
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Risk factors for a high batch-
level bacteriologic prevalence of Y. enterocolitica in Belgian pigs at slaughter. Yersinia 11.
24-28 June 2013, Suzhou, China.
Van Damme, I., Berkvens, D., Vanantwerpen, G., Baré, J., De Zutter, L. Risk factors for
carcass contamination with Yersinia enterocolitica serotype O:3 in Belgian pig
slaughterhouses. Yersinia 11. 24-28 June 2013, Suzhou, China.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Within-batch Prevalence and
Quantification of Human Enteropathogenic Yersinia spp. in Pigs at Slaughter. Eighteenth
Conference on Food Microbiology. Belgian Society for Food Microbiology.12-13
September 2013, Brussels, Belgium.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Seroprevalence of
Enteropathogenic Yersinia spp. in Batches of Pigs at Slaughter. Eighteenth Conference on
Food Microbiology. Belgian Society for Food Microbiology.12-13 September 2013,
Brussels, Belgium.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Risk factors for a high batch-
level bacteriologic prevalence of Y. enterocolitica in Belgian pigs at slaughter. Eighteenth
Conference on Food Microbiology. Belgian Society for Food Microbiology.12-13
September 2013, Brussels, Belgium.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Within-batch Prevalence and
Quantification of Human Enteropathogenic Yersinia spp. in Pigs at Slaughter. ECVPH AGM
and Conference.19-20 September 2013, Turin, Italy.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Seroprevalence of
Enteropathogenic Yersinia spp. in Batches of Pigs at Slaughter. ECVPH AGM and
Conference.19-20 September 2013, Turin, Italy.
CURRICULUM VITAE ________________________________________________________________________
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191
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Risk factors for a high batch-
level bacteriologic prevalence of Y. enterocolitica in Belgian pigs at slaughter. ECVPH AGM
and Conference.19-20 September 2013, Turin, Italy.
2014:
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf, K. Relation between
microbiological and serological data of human pathogenic Y. enterocolitica in pigs and pig
batches. IAFP's European Symposium on Food Safety. 7-9 May 2014, Budapest, Hungary.
Vanantwerpen, G., De Zutter, L., Houf, K. Salmonella: Effect of Chilling Pork on the
Sampling Method. IAFP's European Symposium on Food Safety. 7-9 May 2014, Budapest,
Hungary.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Risk factors for a high batch-
level bacteriologic prevalence of human pathogenic Y. enterocolitica in Belgian pigs at
slaughter. IAFP's European Symposium on Food Safety. 7-9 May 2014, Budapest, Hungary.
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf, K. Relation between
microbiological and serological data of human pathogenic Y. enterocolitica in pigs and pig
batches. sfam Summer Conference. 30 June-3 July 2014, Brighton, U.K.
Vanantwerpen, G., De Zutter, L., Houf, K. Salmonella: Effect of Chilling Pork on the
Sampling Method. sfam Summer Conference. 30 June-3 July 2014, Brighton, U.K.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Risk factors for a high batch-
level bacteriologic prevalence of human pathogenic Y. enterocolitica in Belgian pigs at
slaughter. sfam Summer Conference. 30 June-3 July 2014, Brighton, U.K.
CURRICULUM VITAE ________________________________________________________________________
________________________________________________________________________
192
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf, K. Relation between
microbiological and serological data of human pathogenic Y. enterocolitica in pigs and pig
batches. Food Micro 2014. 1-4 September 2014, Nantes, France.
Vanantwerpen, G., De Zutter, L., Houf, K. Salmonella: Effect of Chilling Pork on the
Sampling Method. Food Micro 2014. 1-4 September 2014, Nantes, France.
Vanantwerpen, G., Houf, K., Van Damme, I., De Zutter, L. Risk factors for a high batch-
level bacteriologic prevalence of human pathogenic Y. enterocolitica in Belgian pigs at
slaughter. Food Micro 2014. 1-4 September 2014, Nantes, France.
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf, K. Relation between
microbiological and serological data of human pathogenic Y. enterocolitica in pigs and pig
batches. Belgian Society for Food Microbiology.18-19 September 2014, Brussels, Belgium.
Vanantwerpen, G., De Zutter, L., Houf, K. Salmonella: Effect of Chilling Pork on the
Sampling Method. Belgian Society for Food Microbiology. 18-19 September 2014,
Brussels, Belgium.
Vanantwerpen, G., Van Damme, I., De Zutter, L., Houf, K. Relation between
microbiological and serological data of human pathogenic Y. enterocolitica in pigs and pig
batches. ECVPH AGM and Conference. 6-8 October 2014, Copenhagen, Denmark.
Vanantwerpen, G., De Zutter, L., Houf, K. Salmonella: Effect of Chilling Pork on the
Sampling Method. ECVPH AGM and Conference. 6-8 October 2014, Copenhagen,
Denmark.
CURRICULUM VITAE ________________________________________________________________________
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193
BUITENLANDSE DIENSTREIZEN
FoodMicro 2012. 3-6 September 2012, Istanbul, Turkey.
Yersinia 11. 24-28 June 2013, Suzhou, China.
ECVPH AGM and Conference.19-20 September 2013, Turin, Italy.
Food Micro 2014. 1-4 September 2014, Nantes, France.
ECVPH AGM and Conference. 6-8 October 2014, Copenhagen, Denmark.
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195
DANKWOORD
DANKWOORD ________________________________________________________________________
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196
Toen ik in mijn derde bachelor diergeneeskunde op zoek was naar een onderwerp voor
mijn Masterproef I, heb ik bij Prof. Dr. Lieven De Zutter op de deur geklopt. Lieven, ik had
nog nooit les van jou gehad en herkende je dus niet toen ik jou de weg naar jouw bureau
vroeg. Sindsdien heb je me altijd geholpen de weg te vinden, doorheen stapels artikels en
hopen CIN-platen. Je hebt me steeds voldoende vrijheid gegeven zodat ik me kon
ontplooien, pistes kon volgen die ik interessant vond en naar congressen kon gaan. Als ik
problemen had tijdens het onderzoek, wist je steeds bij wie ik met vragen terecht kon.
Bedankt ook voor het vertrouwen om een gedeelte van de practica te veranderen en te
herorganiseren. Heel erg bedankt voor alle mogelijkheden dat je me al geboden hebt en
nog steeds biedt.
Prof. Dr. Kurt Houf, wij hebben elkaar pas later echt leren kennen. Toen ik aangenomen
werd als assistent, kwam ik ook onder jouw vleugels terecht. Ik was toen reeds een klein
jaar bezig met mijn doctoraat. Ik kon bij jou steeds terecht voor vragen van moleculaire
aard. Je hebt me geïntroduceerd in de European College of Veterinary Public Health
waarbij je nu mijn Programme Director en Resident Advisor bent. Je hebt me zelfs tot in
het Nederlands Ministerie gebracht. Bedankt voor alles!
Beste juryleden, Prof. Dr. Hristo Najdenski, Dr. Pierre Wattiau, Dr. Lieve Herman, Dr.
Katelijne Dierick, Prof. Dr. Dominiek Maes en Prof. Dr. Frank Pasmans, bedankt voor het
nalezen van dit doctoraatswerk, en voor het geven van opbouwende kritiek die dit werk
zeker opgewaardeerd heeft.
Prof. Dr. Dirk Berkvens, we zijn elkaar meermaals tegengekomen tijdens dit doctoraat, en
altijd kon ik op je rekenen voor statistisch en epidemiologisch getinte problemen. Steeds
heb je me geholpen en me uitgelegd hoe bepaalde problemen aan te pakken en welke
testen te gebruiken. Bedankt voor de tijd, het geduld, en het optimisme wanneer we door
het bos de bomen niet meer zagen.
Vervolgens zou ik graag de slachthuizen willen bedanken. De samenwerking met hen was
altijd zeer aangenaam en ik apprecieer de moeite die jullie gedaan hebben enorm. Telkens
waren jullie bereid om mij toegang te verschaffen in jullie slachthuis. Didier en Niek,
speciale dank naar jullie. Jullie gaven mij steeds de nodige gegevens over slachttijden en
DANKWOORD ________________________________________________________________________
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varkensboeren, of jullie belden zelfs elke keer de varkensboeren op om mij te
introduceren. Soms moest ik niet eens zoeken naar collega’s om me te vergezellen naar
het slachthuis, maar kreeg ik hulp van jullie. Bedankt voor de hulpvaardigheid en de
interesse in dit onderzoek!
Uiteraard zou ik graag ook mijn (ex-)collega’s willen bedanken. Met de collega’s waarmee
ik een bureau deel/gedeeld heb, Anneleen, en later Ellen en Natascha, heb ik talloze
discussies gevoerd over al dan niet werkgerelateerde onderwerpen. Daarnaast mag ik
Sarah, Adelheid, Laïd, Julie, Emily, Inge, Tomasz, Glynnis, Bavo en Francesca niet vergeten,
die steeds klaar stonden om eens mee te gaan naar het slachthuis, om platen te drogen
wanneer ik terug kwam of om goede raad bij het verwerken van data. Ook de vele
geanimeerde discussies zal ik niet vergeten! Natuurlijk verdienen Martine, Carine, Sandra,
Annelies, Sofie en Jeroen ook een woord van dank. Zonder hun praktische hulp en
toewijding in het lab waren het soms enorm lange werkdagen geworden. Ook aan de fijne
momenten naast het werk heb ik veel leuke herinneringen. Nog een extra dankwoordje
voor Soetkin, die steeds klaarstond bij administratieve problemen. Dan zijn er nog Johan
en Lieve, die me ingewijd hebben in het geven van practica. Tot slot zijn er collega’s die
nog niet vermeld zijn: Lynn, Julie, Jella, Lieselot, Lieven, Julie, Kaat, Nathalie, Gabriel,
Mieke, Joke, Ine en Dirk. Bedankt voor de leuke babbels of voor de ambiance in de resto.
Dan zijn er ook nog mijn andere vrienden-doctoraatstudenten. Bedankt voor het
luisterend oor als ik even wilde ventileren, voor de inbreng als ik eens een andere opinie
wilde horen of om samen een cursus te volgen.
De laatste personen die ik wil bedanken, maar daardoor zeker niet de minst belangrijkste,
zijn mijn ouders. Mama en papa, jullie hebben me altijd gesteund tijdens mijn studies.
Jullie staan altijd klaar als ik niet weet hoe iets praktisch aan te pakken of zelfs om eens
mee te gaan naar het slachthuis. Ik kan altijd op jullie rekenen. Bedankt voor jullie
eindeloos geduld als ik eens uitvoerig over dit onderzoek aan het vertellen was.
Gerty
29 september 2014