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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5

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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7

GENERAL INTRODUCTION

GENERAL INTRODUCTION ________________________________________________________________________

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8

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,


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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).


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20

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


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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


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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


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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).


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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).


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25

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


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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).


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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


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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.


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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.


Number of pigs

Positive batches

(%)


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


Number of pigs

Positive batches

(%)


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


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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.


________________________________________________________________________

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


________________________________________________________________________

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


________________________________________________________________________

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


________________________________________________________________________

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


Country


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


Country


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.

________________________________________________________________________

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 ________________________________________________________________________

________________________________________________________________________

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 ________________________________________________________________________

________________________________________________________________________

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 ________________________________________________________________________

________________________________________________________________________

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 ________________________________________________________________________

________________________________________________________________________

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 ________________________________________________________________________

________________________________________________________________________

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.

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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).


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

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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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77

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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78

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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81

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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87

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.


3.1. 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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89


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


0

20

40

60

80

100

120

140

160

num

ber

of

pig

s


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)

CHAPTER 4 ________________________________________________________________________

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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%

CHAPTER 4 ________________________________________________________________________

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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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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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99

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.


3.1. 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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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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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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111

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

(%

)


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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112

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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113

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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117

CHAPTER 6

FACTORS INFLUENCING THE SEROPREVALENCE OF

HUMAN PATHOGENIC YERSINIA SPP. IN

FATTENING PIGS AT SLAUGHTER

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118

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.


3.1. 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

(%)


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

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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


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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).


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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

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e (O

D%

)

Yersinia count (log10 CFU/g tonsil)


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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


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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.


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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


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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

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

SUMMARY ________________________________________________________________________

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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.

SUMMARY ________________________________________________________________________

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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.

SUMMARY ________________________________________________________________________

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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

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).

SAMENVATTING ________________________________________________________________________

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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

SAMENVATTING ________________________________________________________________________

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de serologische prevalentie voor het slachten. De resultaten van deze prevalenties

werden vergeleken via een mixed-effect logistische regressie op dieren 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

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151

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.

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REFERENCES ________________________________________________________________________

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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.


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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.


Pathogenic Yersinia Enterocolitica in Pigs at Slaughter. Seventeenth Conference on Food


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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.


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.


Enteropathogenic Yersinia spp. in Batches of Pigs at Slaughter. Yersinia 11. 24-28 June

2013, Suzhou, China.


Quantification of Human Enteropathogenic Yersinia spp. in Pigs at Slaughter. Yersinia 11.

24-28 June 2013, Suzhou, China.


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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.


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.


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.


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.


Quantification of Human Enteropathogenic Yersinia spp. in Pigs at Slaughter. ECVPH AGM

and Conference.19-20 September 2013, Turin, Italy.


Enteropathogenic Yersinia spp. in Batches of Pigs at Slaughter. ECVPH AGM and

Conference.19-20 September 2013, Turin, Italy.


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191


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.


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.



batches. sfam Summer Conference. 30 June-3 July 2014, Brighton, U.K.


Sampling Method. sfam Summer Conference. 30 June-3 July 2014, Brighton, U.K.



slaughter. sfam Summer Conference. 30 June-3 July 2014, Brighton, U.K.


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192



batches. Food Micro 2014. 1-4 September 2014, Nantes, France.


Sampling Method. Food Micro 2014. 1-4 September 2014, Nantes, France.



slaughter. Food Micro 2014. 1-4 September 2014, Nantes, France.



batches. Belgian Society for Food Microbiology.18-19 September 2014, Brussels, Belgium.


Sampling Method. Belgian Society for Food Microbiology. 18-19 September 2014,

Brussels, Belgium.



batches. ECVPH AGM and Conference. 6-8 October 2014, Copenhagen, Denmark.


Sampling Method. ECVPH AGM and Conference. 6-8 October 2014, Copenhagen,

Denmark.


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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 ________________________________________________________________________

________________________________________________________________________

197

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

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