Department of Reproduction, Obstetrics and Herd Health
Department of Pathology, Bacteriology and Poultry Diseases
Epidemiology of M. hyopneumoniae infections and effect
of control measures
Iris Villarreal
Thesis to obtain the academic degree of Doctor of Veterinary Science (PhD)
Faculty of Veterinary Medicine, Ghent University
2010
Promoters:
Prof. dr. D. Maes; Prof. dr. F. Haesebrouck; Prof. dr. Frank Pasmans
1
The cover photo was taken by Aaron Fortin. All rights reserved.
This thesis was printed thanks to the kind support of Pfizer Animal Health.
2
CONTENTS
CONTENTS
LIST OF ABBREVIATIONS
CHAPTER 1. GENERAL INTRODUCTION
1.1. Literature review
1.1.1. Aetiology and pathogenesis of enzootic pneumonia
1.1.2. Epidemiology of Mycoplasma hyopneumoniae
1.1.3. Symptoms, lesions and diagnostic methods
1.1.4. Host immune response and virulence
1.1.5. Control of Mycoplasma hyopneumoniae
CHAPTER 2. AIMS OF THE STUDY
CHAPTER 3. EXPERIMENTAL STUDIES
Early infection and transmission of Mycoplasma hyopneumoniae under field conditions
3.1 Early Mycoplasma hyopneumoniae infections in suckling pigs in herds with respiratory
problems in Europe: detection rate and risk factors
3.2 Effect of vaccination against Mycoplasma hyopneumoniae on the transmission of M.
hyopneumoniae under field conditions
Protection against different strains of Mycoplasma hyopneumoniae
3.3 Effect of vaccination against experimental infection with highly and low virulent
Mycoplasma hyopneumoniae strains
3.4 Infection with a low virulent Mycoplasma hyopneumoniae isolate does not protect piglets
against subsequent infection with a highly virulent M. hyopneumoniae isolate
CHAPTER 4. GENERAL DISCUSSION
SUMMARY
3
ABBREVIATIONS
LIST OF ABBREVIATIONS
AIAO
ANOVA
ADG
BALF
BALT
CCU
DPI
EP
ELISA
EU
ELI
HPLC-H2O
HV
All-in/ all-out
Analysis of variance
Average daily weight gain
Bronchoalveolar lavage fluid
Bronchus-associated lymphoid tissue
Color changing units
Days post infection
Enzootic pneumonia
Enzyme-linked immunosorbent assay
European Union
Expression library immunization
High performance liquid chromatography water
Highly virulent
H2O2
IF
Ig
IFN-γ
IL
LV
nPCR
NrdF
NV
OD
PBS
PCR
PCV2
PRCV
PRDC
PGE2
RDS
Rn
PRSSV
SPES
SD
S-I
SIV
Th
TNF-α
V
w
Hydrogen peroxide
Immunofluorescence
Immunoglobulin
Interferon gamma
Interleukin
Low virulent
Nested polymerase chain reaction
Ribonucleotide reductase
Non vaccinated
Optical density
Phosphate buffered saline
Polymerase chain reaction
Porcine circovirus type 2
Porcine respiratory corona virus
Porcine respiratory disease complex
Prostaglandin E
Respiratory disease score
Adjusted reproduction Ratio
Porcine reproductive and respiratory syndrome virus
Slaughterhouse Pleurisy Evaluation System
Standard deviation
Susceptible-infectious
Swine influenza virus
T helper cells
Tumor necrosis factor alfa
Vaccinated
Weeks
4
CHAPTER 1: GENERAL INTRODUCTION
CHAPTER 1.
GENERAL INTRODUCTION
5
CHAPTER 1: GENERAL INTRODUCTION
1.1. LITERATURE REVIEW
Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary etiological agent of enzootic
pneumonia (EP), one of the most prevalent respiratory diseases in pigs (Thacker, 2006). In field
situations, it acts as a door opener for secondary bacterial pathogens. M. hyopneumoniae is one
of the main pathogens involved in the porcine respiratory disease complex (PRDC) (Opriessnig
et al., 2004; Thacker, 2006). PRDC is often caused by M. hyopneumoniae, accompanied by
infections with secondary bacteria such as Actinobacillus pleuropneumoniae, Haemophilus
parasuis, Pasteurella multocida, Streptococcus suis, as well as with viral agents such as porcine
reproductive and respiratory syndrome virus (PRSSV), porcine respiratory corona virus (PRCV),
swine influenza virus (SIV), and porcine circovirus type 2 (PCV2). EP and PRDC inflict
worldwide economic losses in the swine industry (Ross, 1999; Rautiainen et al., 2000; Georgakis
et al., 2002; Maes et al., 2008) due to decreased growth rate, increased feed conversion ratio,
increased treatment costs and increased mortality, emphasizing the importance of M.
hyopneumoniae among the scientific community.
This review aims to discuss the most recent findings on the following aspects of M.
hyopneumoniae infections and/or enzootic pneumonia: the aetiology and pathogenesis, the
epidemiology of M. hyopneumoniae infections with special emphasis on transmission,
differences between low and highly virulent M. hyopneumoniae strains, and finally strategies for
control with focus on vaccination.
1.1.1. Aetiology
M. hyopneumoniae belongs to the mollicutes taxon and ranks among the smallest (0.2 µm) selfreplicating bacteria known to date (Tajima and Yagihashi, 1982; Jacques et al., 1992; Blanchard
et al., 1992). It has a very small and simple genome (893 - 920 kilo-base pairs), and the organism
6
CHAPTER 1: GENERAL INTRODUCTION
belongs to the most sequenced porcine mycoplasmas (strains J, 232 and 7448) (Minion et al.,
2004; Vasconcelos et al., 2005). M. hyopneumoniae is extremely sensitive to environmental
conditions and in contrast to most prokaryotic organisms, it is characterized by the lack of a
cellular wall. The pathogen is not able to survive for a long time outside its host, but in aerosols
its survival time increases as it can remain infective for up to 31 days in water at 2-7oC
(Goodwin, 1972).
The organism is characterized by a slow and fastidious growth in vitro and is extremely difficult
to isolate (Friis, 1974). Therefore, isolation is not used for routine diagnosis. The first isolations
of M. hyopneumoniae were done from pneumonic lung tissue and took place only four decades
ago (Goodwin et al., 1965; Maré and Switzer, 1965). The J strain was isolated in 1973
(Whittlestone, 1973). It has become a high passage laboratory strain that is considered as the
reference strain for M. hyopneumoniae. The fastidious growth is related to its dependence on the
supply of nutrients and the need for constant environmental conditions. Therefore, a wide
spectrum of essential amino acids and fatty acids provided by either the host or the culture
medium is required, since a considerable proportion of genes involved in cofactor biosynthesis
has been lost throughout its evolution (Razin et al., 1998; Pollack, 2002).
A parasitic way of life has been achieved through the manifestation of surface proteins
(Chambaud et al., 1999). The organism can produce adhesins, modulins, aggressins and
impedins that allow adhesion and modulation of the host immune system (Asai et al., 1996;
Henderson et al., 1996; Muneta et al., 2006, 2008). Using molecular techniques, it appears that
M. hyopneumoniae isolates show a large diversity at genomic (Stakenborg et al., 2006; Major et
al., 2007; Strait et al., 2008) and proteomic (Calus et al., 2007) level. In addition, there are also
important differences in virulence among M. hyopneumoniae isolates (Vicca et al., 2003; Strait et
7
CHAPTER 1: GENERAL INTRODUCTION
al., 2004). Based on respiratory disease score, lung lesions scores, histopathology,
immunofluorescence and serology, three groups of isolates were obtained, namely low, moderate
and highly virulent ones (Vicca et al., 2003).
1.1.2. Pathogenesis
M. hyopneumoniae is a host specific bacterium which only infects pigs. The organism attaches to
cilia and colonizes the mucosal surface of the ciliated epithelium of the trachea, bronchi and
bronchioles of pigs (Blanchard et al., 1992; Kwon et al., 2002; Saradell et al., 2003). Adhesion is
a prerequisite for initiation of disease (Tajima and Yagihashi, 1982). It is a process mediated by
carbohydrates and proteins (Zielinski et al., 1993; Li et al., 2009) present on this
microorganism‘s surface and the ciliated epithelium of the respiratory tract of the pig. Although
adhesion to cilia is important to cause pneumonia, differences in virulence between strains were
not associated with their adhesion capacity in vitro (Calus et al., 2009). Some Mycoplasma
species (e.g. M. gallisepticum) have the ability to penetrate and live within host cells (Winner et
al., 2000), but there is no such evidence for M. hyopneumoniae (Jenkins et al., 2006).
M. hyopneumoniae infections interfere with the normal functioning of the cilia in the respiratory
tract, affecting the nonspecific defense mechanisms. As a result, infected animals are more
susceptible to secondary bacterial infections (Ciprian et al., 1988; Sørensen et al., 1997). The
ciliated epithelial cells of the trachea, the bronchi and bronchioles, the secretion of mucus and
the alveolar macrophages are important for removing noxious inhaled particles and pathogens
from the respiratory system. Once the ciliary activity is impaired, the mucosal clearance system
becomes ineffective, leading to hypersecretion and evident presence of exudate caused by an
altered glycoprotein production in goblet cells (DeBey et al., 1992). The mechanism behind cilia
8
CHAPTER 1: GENERAL INTRODUCTION
damage is not fully understood, but it has been suggested that an increased inflow of calcium
ions into the epithelial cells (DeBey et al., 1993; Park et al., 2002) may result in ciliostasis and
gradual loss of cilia. A membrane-associated cytopathic factor (54 kDa protein) has been found
to induce cytotoxicity to tracheal rings, but its role remains unclear (DeBey and Ross, 1994). For
some mycoplasmas (e.g. M. mycoides) (Miles et al., 1991; Rice et al., 2001), it has been
suggested that the production of hydrogen peroxide (H2O2) might be involved in the damage of
host cells. It is not known whether this is also the case for M. hyopneumoniae. The damage of the
epithelium in the respiratory tract and the infiltration of exudate often facilitate the entry of other
respiratory pathogens such as P. multocida, A. pleuropneumoniae, B. bronchiseptica, among
many others, that can considerably aggravate the condition of the infected pig.
In addition, the ability of M. hyopneumoniae to modulate the host immune response enables it to
evade or suppress the pig‘s defense mechanisms and establish a chronic, persistent infection
(Razin et al., 1998). Studies have shown that the organism remains in the lungs until at least 6
(Fano et al., 2005a) or 8 months (Pieters et al., 2009) after challenge infection. It has been
demonstrated that phagocytosis by alveolar macrophages is suppressed during M.
hyopneumoniae infection (Caruso and Ross, 1990). Moreover, Asai et al. (1996) demonstrated
that the chemiluminescence response in neutrophils obtained from broncho-alveolar lavage
(BAL) fluid in pigs experimentally infected with M. hyopneumoniae was suppressed, indicating
a decrease in the function of these cells.
The mechanisms involved in the pathogenicity of M. hyopneumoniae remain unclear. A
remarkable increase in the number of neutrophils and higher titres of pro-inflammatory cytokines
TNF-α and IL-1 in BAL fluid were detected in pigs infected with a highly virulent strain
compared to those infected with low virulent ones at 10, 15 and 28 DPI (Meyns et al., 2007). A
9
CHAPTER 1: GENERAL INTRODUCTION
faster in vitro multiplication of the highly virulent isolate during the logarithmic phase has also
been described (Calus et al., 2010), suggesting a higher capacity to multiply in the lungs. Further
research is ongoing to compare the colonization characteristics of highly and low virulent strains.
1.1.3. Occurrence and epidemiology of M. hyopneumoniae infections
M. hyopneumoniae has been detected in almost all pig farms in countries with intensive pig
production (Furlong et al., 1975; Madec and Kobisch, 1982; Rautiainen et al., 2001; Sibila et al.,
2004a; Maes et al., 2008; Silva et al., 2009). Table 1 shows the occurrence of infection in pigs of
different age groups reported in various studies.
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CHAPTER 1: GENERAL INTRODUCTION
Table 1: Occurrence of M. hyopneumoniae in pigs of different ages
Occurrence of M.
hyopneumoniae
Suckling pigs
13.0%
1.5-3.8%
0.5-2.1%
12.3%
Weaned pigs
10.0-60.0%
1.3-13.1%
10.1%
0.0-51.2%
2.8-7.8%
3.0-51.0%
10.6%
Fattening pigs at
slaughter
19.0%
0.0-100%
88.0%
54.0-81.0%
8.0-100%
29.3-61.1%
79.0-86.0%
Sows
24.0-56.0%
27.5%
33.3%
24.0-36.0%
nPCR
(nasal
swabs)
nPCR
(bronchial/
tracheal
swabs)
nPCR
(BALF)
Serology
+
Author (s)
Palzer, 2006
+
+
Sibila et al., 2007a
Sibila et al., 2007b
+
Moorkamp et al., 2009
+
+
+
+
+
Calsamiglia et al., 1999
Ruiz et al., 2003
Sibila et al., 2004
Fano et al., 2007
Sibila et al., 2007b
+
+
Sibila et al., 2008
Moorkamp et al., 2009
+
+
+
+
+
Morris et al., 1995
Maes et al., 1998
Maes et al., 1999
Rautiainen et al., 2000
Fano et al., 2007
+
Sibila et al., 2007b
+
Sibila et al., 2007b
+
Calsamiglia, 2000
+
+
+
65.0%
+
Sibila et al., 2007a
Sibila et al., 2007b
Sibila et al., 2008
grosse Beilage et al.,
2009
Suckling and weaned pigs
The percentage of suckling pigs positive for M. hyopneumoniae by nPCR in BAL fluid among
the suckling population (1-3 weeks of age) in two German studies was approximately 10%
(Moorkamp et al., 2009; Palzer, 2006). A similar but slightly higher percentage of positive pigs
11
CHAPTER 1: GENERAL INTRODUCTION
by nPCR in BAL fluid was found in weaned pigs (3-6 weeks of age) in Germany (Moorkamp et
al., 2009) and Spain (Sibila et al., 2004; Sibila et al., 2008). In all these studies, the selected
herds had a history of respiratory disease. Under field conditions, there is a progressive decrease
in the numbers of pigs with maternal antibodies followed by a slow increase in the numbers of
seropositive animals towards the end of the nursery stage (Calsamiglia et al., 1999b). The
prevalence of M. hyopneumoniae based on nPCR testing on nasal swabs in weaned pigs has been
suggested as a potential indicator of shedding by sows (Ruiz et al., 2003) and a possible predictor
of the severity of enzootic pneumonia in older animals (Fano et al., 2007; Sibila et al., 2007a;
and Sibila et al., 2007b).
Fattening pigs
The percentage of animals with antibodies against M. hyopneumoniae at slaughter ranges from 0
to 100% (Maes, 1998). However, the prevalence largely varies between farms, production
systems and countries. Maes et al. (1999) investigated epidemiological aspects in slaughter pigs
from 50 fattening pig herds, reporting that the seroprevalence at herd level for M.
hyopneumoniae was 88%. Rautiainen et al. (2000) reported that seroprevalences fluctuated
between 54 and 81% in fattening pigs. Detection in bronchial (or tracheal) swabs by nPCR
ranges between 79-86% (Sibila et al., 2007b) and up to 100% (Fano et al., 2007).
Sows
M. hyopneumoniae seroprevalence in sows varies widely between studies namely from 0 to 56%
(Calsamiglia and Tijoan 2000; Sibila et al., 2007). A cross-sectional study carried out in 2578
sows from 67 herds in north-west Germany by grosse Beilage et al. (2009) showed that 65% of
the sows were seropositive.
12
CHAPTER 1: GENERAL INTRODUCTION
Despite the common practice of vaccination and its beneficial effects against EP in pigs, the
overall occurrence of this pathogen is not significantly different in vaccinated and non
vaccinated animals (Meyns et al., 2006; Maes et al., 2008).
1.1.3.1.
Transmission of Mycoplasma hyopneumoniae
Within a herd, M. hyopneumoniae can be transmitted by both vertical and horizontal
transmission (Ross, 1999; Thacker, 2006; Fano et al., 2007). Vertical transmission is the transfer
of a pathogen from a parent (usually the mother) to the offspring. Horizontal transmission is the
transfer of a pathogen from an infected animal to a naive animal, independent of the parental
relationship of those individuals. Horizontal transmission of M. hyopneumoniae may occur by
either direct nose-to-nose contact with infected pigs or indirectly by sharing the same air space
with infected animals. Airborne transmission is considered a form of indirect horizontal
transmission by which pathogenic agents contained in aerosols spread from infected to noninfected susceptible individuals (Kurkjian et al., 2003). Introduction of infected animals and
airborne transmission (Cardona et al., 2005) are the two main sources for introduction of M.
hyopneumoniae in a herd (Ross, 1999).
Sow to piglet transmission
Despite the limited existence of studies on sow-to-piglet transmission, it is generally accepted
that the sow plays an important role in transmitting the pathogen to the offspring, and
consequently, to maintain the infection within a herd (Sibila et al., 2007; grosse Beilage et al.,
2009). Transmission occurs when the sow has direct nose-to-nose contact with her suckling
piglets (Rautiainen and Wallgren, 2001; Thacker et al., 2006), since no in-utero or lactogenic
transmission has been documented (Burgi, 1990; Maes et al., 1998). Sibila et al. (2007)
13
CHAPTER 1: GENERAL INTRODUCTION
demonstrated that M. hyopneumoniae can be detected in the lower and upper respiratory tract of
piglets as early as between the 1st and 3rd week of age.
Several studies have shown that younger sows are more likely to transmit the infection to the
offspring (Goodwin 1965; Morris et al., 1994; Sibila et al., 2007). Fano et al. (2006) showed that
the prevalence at weaning in piglets from young sows (parity 1-2) was 1.6 higher than in piglets
from older sows (parity > 5). Hence, the risk of being infected or tested positive decreases with
parity or age of the sow. Partial depopulation in a herd (removing pigs younger than 10 months
of age and leaving the older sows) has been used as an alternative in the past for a successful
eradication protocol in Switzerland (Zimmermann et al., 1989). The program was shown to be
successful to eliminate M. hyopneumoniae in small pig herds. It is currently seldom used in
larger pig herds.
These findings also correspond with those of a recent cross-sectional study involving 2578 sows
from 67 herds in Germany, in which a negative correlation was found between seropositivity in
sows and their parity (grosse Beilage et al., 2009). Although older sows (third to seventh parity)
are less likely to transmit M. hyopneumoniae to their offspring, they also retain the potential to
spread M. hyopneumoniae to their piglets (Calsamiglia and Pijoan, 2000).
Horizontal transmission
Horizontal transmission can occur by direct nose-to-nose contact (Done, 1996) with respiratory
secretions from infected animals or water droplets from coughing pigs to susceptible animals, or
by indirect airborne transmission (Goodwin, 1985; Stärk et al., 1998a; Dee et al., 2009).
a. Direct contact
14
CHAPTER 1: GENERAL INTRODUCTION
Nose-to-nose transmission can occur from infected to susceptible animals in all age groups.
(Clark et al., 1991). Pigs from different pens can infect each other by direct contact if no solid
pen partitions are present. Based upon findings of an experimental transmission study, pigs
infected with M. hyopneumoniae will infect on average one penmate during the nursery period
(Meyns et al., 2004), indicating that the spread of this pathogen is slow (Batista et al., 2004; Fano
et al., 2005a; Meyns et al., 2006). Piglets between 3 and 12 weeks of age seem to be equally
susceptible to this pathogen (Piffer et al., 1984). Once the pathogen has been introduced and
settled in the herd, it continues to spread to other age groups and infection is maintained within
the farm.
Since parameters like temperature and humidity are influenced by outdoor weather conditions,
infection and reinfection are more likely to occur in autumn and winter (Goodwin et al., 1985;
Jorsal et al., 1988; Thomsen et al., 1992). It has been observed that clinical signs of mycoplasmal
disease in pigs reach their peak between November and March (Stärk, 1992), due to the
pathogen‘s ability to survive in cold and wet conditions. One of the first authors to investigate
the influence of temperature and humidity on enzootic pneumonia was Gordon (1963). He
concluded that warmer environmental conditions had a beneficial effect on respiratory diseases
in pigs. Some years later, Whittlestone (1976) also showed experimental evidence of the effect of
temperature and humidity on the survival of M. hyopneumoniae by demonstrating an inverse
relationship between enzootic pneumonia and temperature increase in dry conditions. Finally,
Geers et al. (1989) observed a negative correlation between coughing and air temperature.
Multi-source purchase policy is considered as one of the most important factors in the
introduction of respiratory agents by infected animals into a farm (Rosendal and Mitchell, 1983;
Thomsen et al., 1992; Hurnik et al., 1994; Stärk, 2000; Rautiainen, 2001). Hege et al. (2002)
15
CHAPTER 1: GENERAL INTRODUCTION
reported that pig farmers who bought animals from only one supplier per batch had a
significantly lower risk for introduction of this pathogen than farmers with other purchasing
practices.
On the other hand, littermates can retain the pathogen in their lungs without displaying clinical
symptoms (Goodwin, 1965). Consequently, the likelihood of introduction of infected animals
increases when purchased pigs remain carriers of the organism without displaying symptoms.
Infected animals may carry M. hyopneumoniae bacteria in the lungs even up to six months post
infection (Fano et al., 2005a).
The transmission of two different M. hyopneumoniae strains in nursery piglets was first
quantified by estimating a transmission ratio six years ago (Meyns et al., 2004). In that
experimental study, infected seeder pigs were placed together with susceptible animals during 6
weeks and the number of contact-infected animals was determined at the end of the study, using
Rn-values. The transmission ratios (Rn-value)s of the highly virulent (1.47) and the low virulent
isolates (0.85) were not significantly different, whereas the overall Rn was estimated to be 1.16.
It was concluded that one M. hyopneumoniae infected animal at weaning will infect on average
one penmate during the nursery period.
b. Indirect contact
o Airborne Transmission
Regarding airborne transmission, not only on-farm infected pigs can be a source of infection, but
also infected pigs from neighboring herds. The first successful detection of airborne M.
hyopneumoniae under experimental and field conditions with a PCR assay was performed in
Switzerland (Stärk et al., 1998a).
16
CHAPTER 1: GENERAL INTRODUCTION
The distance to the closest infected farm, its size and the pig density are important risk factors for
infection with M. hyopneumoniae (Dohoo et al., 2009; Stärk, 2000). The risk factors for infection
with this pathogen are presented in Table 2, p.32. In diseased herds, sneezing and coughing
animals generate large amounts of airborne particles, which can potentially cause infection in
susceptible animals. According to Hirst (1995), aerosols are defined as solid or liquid particles
suspended in air. The concentration of infectious agents in aerosols is proportional to the number
and concentration of infected animals on a farm (stocking density, herd size) or a region (pig
density) (Stärk et al., 1998b).
Goodwin (1985) stated that 3.2 km is the minimal prudent distance at which non infected herds
should be situated from M. hyopneumoniae positive herds. Vicinity to infected herds within 2 to
3 km is a risk factor for the aerogen transmission between herds (Thomson et al., 1992).
However, more detailed studies need to be performed in this regard, since the most recent data
have shown that long-distance airborne transmission of this pathogen can occur at a much longer
distance (Dee et al., 2009). M. hyopneumoniae-positive samples have been recovered from air
samples up to 9.2 km from the herd of origin (Otake et al., 2010), and these samples were still
infectious.
The influence of environmental conditions and seasonal patterns also seem to have an effect on
the airborne spread of M. hyopneumoniae (see Table 2, p. 32), although the causal relationship
between environmental risk factors and respiratory diseases is rather complex (Stärk, 2000).
The details of airborne survival for many bacterial species have not been studied in detail. It is
known, however, that radiation, sun light, air ions, relative humidity and pollutants affect the
biological decay of aerosolised agents. The sensitivity to some of these factors is related to the
lipid surface composition of a microorganism. M. hyopneumoniae and other mycoplasmas have a
17
CHAPTER 1: GENERAL INTRODUCTION
large number of prominent lipoproteins on the surface membrane (Chambaud et al., 1999;
Ferreira et al., 2007), which appears to have a marked influence on their survival at low
temperatures (Raccach et al., 1975). In fact, micro-organisms generally survive better at low
temperatures (Heber et al., 1988; Butera et al., 1991). Such resistance to open air factors is
important for this pathogen to remain infective, even when spread over long-distances.
A study about infectious aerosols in pig disease transmission (Stärk, 1998b) states that long
distance transport and survival of airborne microorganisms in pigs seems to be favored by damp,
cool, dark conditions, and in areas with flat topography (free of vegetation) and stable
atmospheric conditions. All these aforementioned conditions seem to favor long distance
transmission of M. hypneumoniae.
o Mechanical vectors
The role of personnel and fomites as mechanical vectors for the transmission of M.
hyopneumoniae between pigs or between farms has not been clearly determined (Done, 1996;
Amass et al., 2000; Batista et al., 2004) and is considered to be of limited importance. Hege et al.
(2002) reported that a parking site for pig transport vehicles situated 10 to 200 m to the farm was
one of the main risk factors for re-infection of herds with M. hyopneumoniae. In contrast to these
findings, Batista et al. (2004) reported that fomites were not important for the transmission of M.
hyopneumoniae. Further studies are necessary to clarify the role of equipment and personnel in
the transmission of M hyopneumoniae.
18
CHAPTER 1: GENERAL INTRODUCTION
1.1.4. Risk factors for infection with M. hyopneumoniae in pig herds
The most important risk factors for infection with M. hyopneumoniae are related to herd
characteristics, management practices and housing conditions, biosecurity measures and seasonal
effects. A list of the major risk factors associated with the risk for M. hyopneumoniae infections
and / or respiratory disease and published in literature, is presented in the control section of this
introduction (see further Table 2, p. 32).
Herd size and herd type are factors among herd characteristics found to influence the incidence
of enzootic pneumonia in pigs (Goodwin et al., 1985; Stark et al., 2000; Maes et al., 2000). In
general, there is a positive association between increasing herd size and the presence of M.
hyopneumoniae infections in the herd.
Different purchase policies are also reflected in different herd types. For instance, growingfinishing farms are more dependent on animal supply than breeding farms are, which makes
them more likely to buy large numbers of animals from different sources (Hofer et al., 2002;
Stark et al., 2000). A multi-source purchase policy largely contributes to introduction of
clinically or sub-clinically infected animals that can easily spread the pathogen among
susceptible pigs (Thomsen et al. 1992; Hege et al., 2002).
The clinical outcome of M. hyopneumoniae infection is also dependent on environmental and
management conditions and on the production system in operation. For instance, a continuous
flow of pigs through facilities increases the odds of infection. Production systems where pigs are
moved through the different production stages in batches (all-in/all-out), as opposed to
continuous flow systems, generally include cleaning and disinfecting steps which reduce microorganism concentrations (Stark et al., 2000). In most reviewed papers, the all-in/ all-out system
was highly advantageous in terms of respiratory disease reduction (Clark et al. 1991; Stark et al.,
19
CHAPTER 1: GENERAL INTRODUCTION
1998, Diekman et al., 1999; Maes et al., 1996). High stocking density (Clark et al., 1991; Hurnik
et al., 1994; Stark et al., 1998b; 2000) is also a decisive factor in increasing the infection pressure
and maintaining respiratory pathogens in the herd.
Biosecurity measures should not be underestimated when control of enzootic pneumonia is
concerned. Hege et al. (2002) found that the presence of parking sites for pig transport at a
distance of 200m or less from the farm constitutes a potential source for introduction of
respiratory diseases. Environmental variables also influence the presence of M. hyopneumoniae
infections in the farm. Low sunlight levels (Dee et al., 2009), low relative air humidity (Stark et
al., 1998a) and low temperature levels, particularly during autumn and winter (Gordon, 1963;
Whittlestone, 1976; Goodwin, 1985; Jorsal et al., 1988; Maes et al., 2001) appear to be strongly
correlated with the presence of this pathogen in pigs. When considering airborne transmission,
not only infected on-farm animals act as infection sources, but also nearby herds with M.
hyopneumoniae infection and/or respiratory problems. The presence of infected neighbouring
farms (Goodwin, 1985; Stark et al., 1992; Hege et al., 2002; Dohoo et al., 2009) and the pig
density in the area (Rose et al., 2002; Zhuang et al., 2002) can put herds free of M.
hyopnemoniae at risk of getting infected.
To effectively control M. hyopneumoniae infections in pigs, it is important to eliminate or reduce
the effects of each of these risk factors (see chapter 1.1.7).
20
CHAPTER 1: GENERAL INTRODUCTION
1.1.5. Symptoms, lesions and diagnostic methods
Clinical signs and lesions
Infection with M. hyopneumoniae often appears to have a subclinical course (Clark et al., 1991).
However, experimental infection with M. hyopneumoniae may lead to coughing, which can be
preceded by a slight increase in body temperature. Coughing usually begins approximately two
weeks after challenge, reaches its peak after five weeks and declines gradually (Whittlestone et
al., 1972; Kobisch et al., 1993). Infections also lead to decreases in average daily weight gain
and to increased feed conversion ratio (Maes et al., 1999; Sibila et al., 2009).
Infection leads to consolidation of the lung tissue and catarrhal pneumonia with purple to grey
regions of meaty aspect with exudate in the airways (Kobisch et al., 1993). Such consolidation is
visible as gross lesions, which are generally observed from 3-12 weeks post infection. The
lesions are mainly localized in the apical and cardial lobes, as well as in the anterior part of the
diaphragmatic lobes and in the intermediate lobe. Lesions resolve after 12 to 14 weeks with
formation of interlobular fissures (Goodwin et al., 1965; Morrison et al., 1985; Blanchard et al.,
1992). Complicated infections with e.g. secondary bacteria may extend the healing period.
Whittlestone (1972) described the histological changes in the lungs, consisting of neutrophil and
lymphocyte infiltration in the lumina, around the airways as well as in the alveoli. As the disease
progresses, these cells increase in number in the perivascular, peribronchial and peribronchiolar
tissue, forming hyperplastic lymphoid nodules (cuffing) around the airways and leading to
alveolar emphysema.
Diagnostic methods
21
CHAPTER 1: GENERAL INTRODUCTION
Clinical signs and lung lesions can be used as an indication of enzootic pneumonia. The severity
of coughing can be assessed using a respiratory disease score (RDS). The system as described by
Halbur et al. (1996) is commonly used and values can range from 0 (no coughing) to 6 (very
severe coughing). In addition, post-mortem inspection of lung lesions in pigs at slaughter age
(Hurnik et al., 1992; Morris et al., 1995; Sibila et al., 2009) is frequently used to investigate the
involvement of M. hyopneumoniae in porcine respiratory disease at the herd level. Scoring
systems to evaluate respiratory disease based on gross pathological lung lesions have been
described by several authors (Hannan et al., 1982; Morrison et al., 1985; Lium and Falk 1991;
Hurnik et al., 1993; Ostanello et al., 2007).
In most pig farms with respiratory problems, 20-80% of the pigs at slaughter display pneumonia
lesions associated with mycoplasmal infection (Morris et al., 1995; Leneveu et al., 2005; Sibila
et al., 2007; Jirawattanapong et al., 2010).
However, assessing clinical symptoms and lung lesion severity are not pathognomonic for
diagnosing enzootic pneumonia (Thacker et al., 2001). More accurate diagnostic methods to
detect the presence of M. hyopneumoniae are necessary to establish a conclusive diagnosis.
Detection of the organism
Bacteriological culture is still the golden standard to identify this organism, but it is seldomly
used due to the slow, impractical and laborious process involved (Thacker, 2004; Sibila et al.,
2009). Either direct or indirect immunofluorescence testing can be performed on lung tissue, but
this cannot be done in life animals (Bradbury, 1998).
Accurate detection of M. hyopneumoniae has significantly increased with development of
polymerase chain reaction (PCR) assays. The first test to specifically amplify and detect M.
22
CHAPTER 1: GENERAL INTRODUCTION
hyopneumoniae DNA was described by Harasawa et al. (1991). Since then, several PCR tests
have been reported (Mattson et al., 1995; Stärk et al., 1998b; Calsamiglia et al., 1999; Caron et
al., 2000; Verdin et al., 2000; Kurth et al., 2002; Thacker et al., 2004; Strait et al., 2008). Nested
polymerase chain reaction (nPCR) is a useful diagnostic tool developed to enhance the detection
threshold of the conventional single-step PCR test (Calsamiglia et al., 2000, Stärk et al., 1998a;
Verdin et al., 2000). The advantages of nPCR are its higher sensitivity and specificity. Nested
PCR is sufficiently sensitive to detect 1-4 organisms / μl of PCR sample (Kurth et al., 2002;
Gebruers et al., 2008). There is however a higher risk for contamination due to repeated pipetting
procedures. To avoid false positive results due to contamination, positive and negative controls
should be used during DNA extraction and amplification. Lung tissue and BAL fluid appear to
be among the most reliable collection sites, especially to detect this pathogen in individual
animals (Kurt et al., 2002). Detection of the organism in samples from the nasal cavity appears
more variable and is more suitable for detection of M. hyopneumoniae at herd level (Otagiri et
al., 2005).
Detection of antibodies
Serology is the most common assay used for detection of serum antibodies against M.
hyopneumoniae at a herd level (Calsamiglia, 1999b; Thacker, 2004). It detects the onset of
seroconversion, not the onset of infection. However, not all animals seroconvert at the same
time. The detection of serum antibodies after experimental challenge has been reported to range
between 2- 6 weeks in nursery pigs (Calsamiglia et al., 1999b). Seroconversion can occur as
early as 7- 9 days PI in individual animals (Sheldrake et al., 1990; Sørensen et al., 1997), but it
takes 3-6 weeks for all animals to seroconvert in a challenged group (Kobisch et al., 1993; Le
Pontier et al., 1994; Leon et al., 2000). In most trials, earlier seroconversion to M.
23
CHAPTER 1: GENERAL INTRODUCTION
hyopneumoniae is observed in vaccinated groups compared with non-vaccinated pigs (Sibila et
al., 2007). However, there are some limitations when using serological tests, as these can fail due
to insufficient sensitivity (Thacker et al., 2006), especially if the infection level in the herd is
low. Specificity can be also reduced due to cross-reactions with other species of mycoplasmas
such as M. floculare and M. hyorhinis (Strasser et al., 1992). Nonetheless, the DAKO® M. hyo
ELISA is a blocking ELISA based on monoclonal antibodies against the 74 kDa protein of M.
hyopneumoniae (Feld et al., 1992) with higher sensitivity than previous ELISA tests, preventing
cross-reactions with M. flocculare and M. hyorhinis.
1.1.6. Immune response
Up to date, detailed information about the immune response following M. hyopneumoniae
infection in pigs is not complete. Following adherence to the pig‘s respiratory tract, a series of
complex interactions takes place. The host immune system responds with both non-specific and
specific defense mechanisms. The non-specific mechanisms include the ‗mucociliary blanket‘
acting as the first barrier in the respiratory tract, cytotoxicity of macrophages, increase of T-cell
and natural killer cells, as well as the enhancement of the expression of cell receptors that
activate the complement cascade (Taylor, 1979; Vicca, 2005a).
The specific response involves systemic and local antibody production, cell-mediated immune
stimulation, opsonization and phagocytosis. However, the immune response does not only help
in the protection against M. hyopneumoniae, but it has also been described to play a role in the
exacerbation of lesions following EP (Suter et al., 1985; Turek et al., 1996; Razin et al., 1998;
Choi et al., 2006).
24
CHAPTER 1: GENERAL INTRODUCTION
After infection with M. hyopneumoniae, there is mainly lymphoid cell infiltration around the
airways, which is the result of chemoattraction induced by the release of immunogenic proteins
and mediators during mycoplasmal infection (Messier et al., 1990; Sarradell et al., 2003). The
lymphoid hyperplasia of the bronchus-associated lymphoid tissue (BALT) progressively leads to
obliteration of the bronchiole‘s lumen and atelectasis of the alveoli (Baskerville et al., 1972;
Choi et al., 2006; Redondo et al., 2009). Alveolar macrophages have a very important role in the
initiation of this massive lymphoid infiltration, which starts being histologically visible as soon
as 7 days after infection (Choi et al., 2006).
An in vitro study demonstrated that alveolar macrophages have increased production of IL-1 and
IL-6 following M. hyopneumoniae infection (Thanawongnuwech et al., 2001). The expression of
pro-inflammatory cytokines such as IL-1α, Il-1β, IL-2, IL-4, IL-6, IL8, IL-10, TNF-α and IFN-γ
has been reported during infection with this pathogen (Asai et al., 1994; Thanawongnuwech et
al., 2003, 2004; Rodriguez et al., 2004; Choi et al., 2006; Lorenzo et al., 2006; Redondo et al.,
2009), exerting an effect on Th1 and Th2 response in the respiratory tract of the infected pigs.
Helper T-cells (Th) are an important component in the response to mycoplasma respiratory
disease and are the most numerous subset in the lymphoid infiltration. Although less numerous,
cytotoxic T-cells are also observed in lungs with M. hyopneumoniae- induced lesions (Sarradell
et al., 2003). Specifically, IL-1, Il-2, IL-4, IL-6 and TNF-α play a role in lymphoid activation
(Baarsch et al., 1995; Murtaugh and Foss, 2002) and exert non specific mitogenic activity
(Messier et al., 1990). IL-2 and IL-8 mobilize the immune system in response to infection
(Redondo et al., 2009). Upon activation, Th1-cells are responsible for activating and increasing
phagocytic and cytotoxic activities of macrophages (Messier et al., 1990; Asai et al., 1994). To a
certain extent, the presence of T-cells and overproduction of certain cytokines such as IL-1 and
25
CHAPTER 1: GENERAL INTRODUCTION
IFN-γ exacerbate the host mediated tissue damage. Consequently, the pathogenesis of
mycoplasmal pneumonia is not only dependent upon direct damage caused by mycoplasma (such
as ciliostasis and exfoliation of epithelial cells), but also damage caused by the immune response
of the pig itself (Choi et al., 2006). Th1-cells have also been shown to activate B cells to produce
opsonizing antibodies such as IgG2 and IgG2b in mice. However, it is the function of Th2-cells
to lead B cells to proliferation and antibody production (Chen et al., 2003). As a consequence, a
particular increase in the level of IgG and IgA is found in the tracheobronchial secretions, lungs
and serum of naturally or experimentally infected pigs (Suter et al., 1985; Messier et al., 1990;
Rodriguez et al., 2004; Redondo et al., 2009). IgG participates in opsonization and phagocytosis
by alveolar macrophages, whereas IgA seems important in local immunity (Walker et al., 1996;
Sarradell et al., 2003). Animals with the lowest M. hyopneumoniae specific antibody levels had
the highest macroscopic lung lesion score (Meyns et al., 2006). Also, less pronounced lesions
and reduced detection of this microorganism occurred when high amounts of immunoglobulin
positive cells were detected in the lungs (Messier et al., 1990).
Results based on detection of Ab should be interpreted carefully as serum Ab measured with
commercial ELISAs are generally accepted not to be protective (Haesebrouck et al., 2004).
Despite the immune mechanisms of the host, this bacterium is still able to persist in the
respiratory tract of the pig. Although lifelong persistence has not (yet) been determined,
immunomodulation may enable M. hyopneumoniae to evade or suppress the pig‘s defense
mechanisms and establish a chronic infection (Razin et al., 1998). An in vivo experiment showed
that convalescent carriers of this pathogen can remain infectious for up to 214 days (Pieters et al.,
2009). It has been observed that phagocytosis by alveolar macrophages is suppressed during M.
hyopneumoniae infection (Caruso and Ross, 1990) and to a greater extent, this
26
CHAPTER 1: GENERAL INTRODUCTION
immunosuppression is correlated to a high level of PGE2 in the BAL fluid of infected pigs (Asai
et al., 1996). Elevated concentrations of IL-10 and IFN-γ during mycoplasmal infection have
also been found to contribute to the inhibition of the phagocytic function of macrophages
(Muneta et al., 2008; Redondo et al., 2009).
In addition, it has been suggested that a combination of mechanisms, such as the slow growing
rate, the capacity to evade the immune response and the ability of M. hyopneumoniae to colonize
cell surfaces (without actual penetration), may prolong the stay of this bacterium within the pig‘s
respiratory system and hamper its total clearance from the respiratory tract.
Newborn pigs also receive passive immunity by colostrum. Apart from antibodies, colostrum
also contains cellular immunity. Colostral lymphocytes are passively transferred from the sows
to their offspring (Bandrick et al., 2008). These cells are considered to play a role in the
development of the immune response of the piglet. Rautiainen and Wallgren (2001) showed that
colostral immunity reduced the severity of lung lesions and the infection rate in young animals
(Martelli et al., 2006), but further research is necessary to assess its precise role. Although
colostral immunity does not prevent piglet colonization (Thacker et al., 2000; Sibila et al., 2008),
a lower seroprevalence at weaning and reduced lung lesions scores at slaughter were observed in
pigs originating from sows with a higher concentration of colostral antibodies (Sibila et al.,
2008).
27
CHAPTER 1: GENERAL INTRODUCTION
1.1.7. Control of M. hyopneumoniae infection
Control of enzootic pneumonia can be accomplished through a number of measures that include
optimization of herd management and housing conditions (Maes et al., 2008), antimicrobial
medication and/or vaccination (Bousquet et al., 1998; Vicca, 2005; Moreau et al., 2004; Martelli
et al., 2006; Maes et al., 2008).
1.1.7.1.
Herd management and housing conditions
Numerous factors that not only have an impact on the clinical course of enzootic pneumonia, but
also on the transmission of M. hyopneumoniae have been extensively described by different
authors throughout the years (Goodwin, 1985; Thomsen et al., 1992; Hurnik et al., 1994; Stärk
1998b; Stärk, 2000; Maes et al., 2008). Table 2 presents some important management and
housing conditions that may enhance the spread M. hyopneumoniae infection in a pig herd.
Optimization of herd management practices
Improvement of the herd management practices is primordial in the control of M.
hyopneumoniae infections and should be the first to be accomplished. Instituting management
changes that reduce the possibilities of spreading M. hyopneumoniae or result in decreased lung
damage by other pathogens may lead to considerable improvement in the control of enzootic
pneumonia. However, also additional factors different from housing and management
conditions, such as strain differences, may determine the infection pattern and clinical course of
the disease (Vicca et al., 2002). A review on the influence of environmental and management
factors on respiratory disease in pigs, including M. hyopneumoniae infections, has been
published by Stärk et al. (2000).
Production system
28
CHAPTER 1: GENERAL INTRODUCTION
All-in, all-out (AIAO) production is probably the most important factor in the control of
enzootic pneumonia since it can interrupt the cycle of pathogen transmissions from older to
younger pigs. It allows the producer to tailor environmental conditions to a uniform population
of pigs and to clean the facilities between groups of pigs. AIAO production also results in better
performance and less lung lesions in slaughter pigs (Ice et al., 1997). Mixing or sorting pigs is a
source of stress to the animals and it increases the probabilities of disease transmission (Clark et
al., 1991). Therefore, an AIAO system in which the same pigs are moved as a group through the
different production stages is to be preferred compared to one where pigs are regrouped during
transfer from one unit to another.
Early weaning
Early weaning (< 3 weeks) can reduce transmission of M. hyopneumoniae organisms from the
sow to the offspring (Harris et al., 1990; Dee, 1994), but it is not allowed to be applied
systematically in the EU. Parity segregation has been used in large production systems as a
means to control several diseases in the breeding herd, including M. hyopneumoniae infections.
Gilts and also their offspring are kept separated from the sows until they reach their second
gestation. By that time, they are expected to have acquired the desired immune status and to
pose no destabilization risk for the herd anymore.
Purchase of animals
Closed pig herds or production systems have a more stable herd immunity compared to herds
where (breeding) pigs are purchased. The risk for destabilization of the herd immunity especially
increases in case purchasing of animals is performed at a regular basis (Hurnik et al., 1994b),
when replacement rates are high, or in case the animals originate from different sources
29
CHAPTER 1: GENERAL INTRODUCTION
(Thomsen et al., 1992). However, the pressure to improve genetics in high performing pig herds
forces many pig producers to purchase their breeding stock. In these cases, it is important to
evaluate the health status of the herd of origin. Ideally, incoming gilts or any replacements
should be of similar or higher health status than the recipient herd to prevent them from
introducing infections not present in the recipient herd. A quarantine or adaptation period of at
least 4 weeks should be respected.
Animal stocking density
Decreasing animal density during the different production stages has been shown to reduce the
level of respiratory disease (Flesja and Solberg, 1981; Hurnik et al., 1994a). Crowding may lead
to increased transmission of pathogens, and to stress reactions, making the pigs more susceptible
to infectious diseases. Too low stocking densities are financially not justified. Therefore, it is
important to find a reasonable compromise between stocking densities that are appropriate for
the health of the pigs and those that maximize the returns on the building's cost. In general, the
stocking density for finishing pigs on fully slatted floors should equal or exceed 0.7 m² per pig
(Lebret et al., 2006).
Prevention of other diseases
Appropriate parasite control measures are necessary to avoid any lung damage caused by
migrating Ascaris suum larvae (Jolie et al., 1998). Lung damage may also arise from infections
with other respiratory pathogens including bacteria and viruses. PRRSV, influenza viruses,
PCV-2 and PRCV, in combination with M. hyopneumoniae, are considered to play an important
role in the development of PRDC (Thacker et al., 2002; Bochev, 2008). The impact of these
30
CHAPTER 1: GENERAL INTRODUCTION
infections can be reduced by means of medication, vaccination, improvement of housing
conditions and implementation of biosecurity measures.
Biosecurity measures
Spread of M. hyopneumoniae within sites and within herds can be controlled by applying basic
biosecurity measures. Some of these measures (concerning purchasing policy, prevention of
other diseases, etc) have been already mentioned. But these measures should also encompass
appropriate hygienic conditions for rearing pigs, surveillance, disease outbreak control and
prophylactic measures (Amass et al., 1999). Also personnel, vehicles for transport, feed, water,
bedding and equipment should be separated by production stages and should not come in contact
with animals of different ages (Hege et al., 2002; Roman et al., 2006; Stankovic et al., 2010). A
biosecurity plan should take into account epidemiological situations, and potential threats to the
herd health and production, as well as possible solutions.
Improvement of housing conditions
Housing and/or environmental changes that optimize the climate of the pigs' environment are
important in the control of M. hyopneumoniae infections. Special attention should be paid to the
temperature set points, fan staging, air inlet and curtain settings, sensor placement, heater
capacity, drafts and building maintenance (Heinonen et al., 2001; Maes et al., 2008; Mul et al.,
2010). However, making environmental changes for improving the climate in inappropriate or
old barns frequently entail extensive remodeling, and therefore, they may be difficult and
expensive to institute and maintain.
31
CHAPTER 1: GENERAL INTRODUCTION
Table 2: Risk factors influencing infection with M. hyopneumoniae in a pig herd
Herd Factors
Increasing herd size
Author (s)
Goodwin, 1985; Stark et al., 1992; Stärk
et al., 1998b; Maes et al., 2000; Stärk,
2000
Clark et al., 1991; Hofer, 1993; Hege et
al., 2002; Maes et al., 2008
Herd type: growing-finishing farm
Housing and Management
Increasing stocking density in the finishing unit
Continuous production system
Multi-source purchase
Purchase of gilts
Parking site for pigs transport vehicles closer than 200m to
the farm
Weather variables
Low temperature (autumn and winter)
Low sunlight intensity
Low air humidity
Others
Neighboring farms infected with M. hyopneumoniae
1.1.7.2.
Done et al., 1991; Hurnik et al., 1994;
Stärk et al., 1992; Stärk et al., 1998b;
Clark et al., 1991; Stärk et al., 1998b;
Diekman et al., 1999
Thomsen et al., 1992; Stärk, 2000; Hege
et al., 2002
Maes et al., 2000
Hege et al., 2002
Gordon, 1963; Whittlestone, 1976;
Goodwin, 1985; Jorsal et al., 1988;
Thomsen et al., 1992; Maes et al., 2001
Dee et al., 2009
Dee et al., 2009
Stark et al., 1998a
Goodwin, 1985; Stärk et al., 1992;
Thomson et al., 1992; Hege et al., 2002;
Rose et al., 2002; Zhuang et al., 2002;
Dohoo et al., 2009
Antimicrobial medication
The lack of a cell wall makes this organism insensitive to the group of antibiotics belonging to βlactam group, such as penicillins and celophalosporins (Braun, 1970; Wu et al., 1997).
Macrolides, tetracyclines and lincosamides include the most commonly used antibiotics in the
control of respiratory diseases in pigs (Timmerman et al., 2006) and have been described to be
also effective against mycoplasma. The use of antibiotics in feed and water is common practice,
which efficacy is often related to the reduction of clinical symptoms, lung lesions, improvement
32
CHAPTER 1: GENERAL INTRODUCTION
of performance parameters and prevention of secondary infections (Stipkovitz et al., 2001; Vicca
et al., 2005; Thacker et al., 2006). Although antibiotic treatments are usually able to control the
disease (Bargen et al., 2004; McKelvie et al., 2005), persistence is observed under field
conditions (Ross, 1999) and experimental infections (Kobisch et al., 1996; Hannan et al., 1997;
Le Carreau et al., 2006).
During critical stages in production, strategic medication can be used. However, the preventive
or strategic use of antimicrobials should be discouraged since it increases the risk of
antimicrobial resistance and in case of inappropriate use, may also lead to antimicrobial residues
in the pigs carcasses at the moment of slaughter (Maes et al., 2008). Acquired resistance of M.
hyopneumoniae under experimental conditions has been documented to tetracyclines and some
macrolides (Inamoto et al., 1994; Hannan et al., 1997), and lately also to lincosamides and
fluoroquinolones (Vicca et al., 2004; 2007). The advantages of using antimicrobials compared to
vaccination include flexibility in their usage and easy administration with feed or water, which
makes them not so labour intensive. Despite the success of medication programmes in the
control of M. hyopneumoniae infections described by various studies (Jouglar et al., 1993;
Walter et al., 2000), often only partial prevention is achieved and sometimes results are
inconsistent since outbreaks may reappear after ceasing treatment (Wallgren et al., 1993;
Thacker et al., 2006).
1.1.7.3.
Vaccination against Mycoplasma hyopneumoniae
Vaccination with inactivated, adjuvanted whole-cell bacterins (alone or in combination with
antibiotics) is frequently used worldwide to control M. hyopneumoniae infections (Erlandson et
al., 2002).
33
CHAPTER 1: GENERAL INTRODUCTION
1.1.7.3.1.
Vaccination strategies
Numerous trials have proven the efficacy of vaccination against M. hyopneumoniae to reduce
clinical disease and Mycoplasma lung lesions and to increase performance parameters of the pigs
(Jensen et al., 2002; Maes et al., 2003; 2008).
It has been also suggested that vaccination decreases the infection level in the herd (Sibila et al.,
2007) and although vaccination does not offer complete protection nor prevents pigs from
becoming infected (Mateusen et al., 2001; 2002; Meyns et al., 2004), vaccination may reduce the
number of Mycoplasma organisms in the respiratory tract of the pig (Meyns et al., 2006). Despite
the fact that vaccination against M. hyopneumoniae is widely practiced, many aspects about its
effect on transmission of the pathogen, effect on different strains and on the immunological
response remain unclear and need to be further investigated.
Different vaccination strategies have been adopted depending on the needs of a particular herd
and on variables such as the production system, type of herd, infection pattern and preferences of
the pig producer. The decision on whether to vaccinate or not depends on the economic
feasibility in the farm and the profits that vaccination will bring in relation to the economic
losses caused by the pig‘s decreased performance. In other words, vaccination not only has to be
efficacious, but also cost-effective (Maes et al., 2003; 2008).
Generally, in Europe, piglets are vaccinated at or before weaning. Vaccination is mostly effective
if active immunity is established before infection, making this a critical factor in the
implementation of vaccination strategies. Initially, the most frequently used vaccines were
double-shot bacterins administered intramuscularly in the first week of life, and repeated 2-3
34
CHAPTER 1: GENERAL INTRODUCTION
weeks later. Notwithstanding their current availability, single-shot M. hyopneumoniae vaccines
have become more popular due to their reduced labor costs and less stress inflicted to the piglets
(Baccaro et al., 2006). There are still some debates as for which vaccination program is more
appropriate. For example, in the US, single dose vaccines are recommended for herds with low
infection pressure and where an all-in/all-out production is practiced (Yeske, 2001). However,
several studies have shown that both single and double shot vaccination programs have similar
protective effects (Morris et al., 2001; Roof et al., 2001; Haesebrouck et al., 2004). Some authors
suggest that the efficacy of vaccination in young piglets may depend on the level of maternally
derived antibodies at the time of implementing vaccination (grosse Beilage et al., 2005; Martelli
et al., 2006; Bandrick et al., 2008). The role of maternal cellular immunity and antibody
mediated immunity in the protection of newborn piglets is not entirely clear and the potential
interference with active immunity needs to be better understood. Despite the discrepancies in
literature and ongoing debate on whether maternal antibodies interfere or not with vaccination
efficacy, almost all pigs in Europe are vaccinated in presence of maternal antibodies with very
good results (Martelli et al., 2006; Maes et al., 2008).
Apart from the active immunization of piglets, also vaccination of gestating sows can be done.
However, the question of whether to vaccinate sows before farrowing or not, remains
unanswered. In practice, sow vaccination is rarely practiced and the main strategy to control M.
hyopneumoniae infection is based on piglet vaccination. (Haesebrouck, 2004). The main purpose
of sow vaccination is to decrease the spread of M. hyopneumoniae from mother to offspring and
to ensure protection of newborn piglets through the passage of maternally derived antibodies.
Some studies have shown that sow vaccination before farrowing can lower infection pressure
and have an effect on vertical transmission, resulting in lower piglet colonization by M.
35
CHAPTER 1: GENERAL INTRODUCTION
hyopneumoniae (Ruiz et al., 2003). However, colostral immunity does not prevent colonization
of the piglets with M. hyopneumoniae.
Sow vaccination protocols are especially recommended as a biosecurity measure in herds
purchasing gilts from M. hyopneumoniae free farms. Another reason of gilt vaccination at the
moment of introduction in the breeding population is to prevent them from destabilizing the
infection level in the herd or as a response to an outbreak of enzootic pneumonia in the farm
(Bargen et al., 2004).
1.1.7.3.2.
Effect of vaccination on transmission of M. hyopneumoniae
Meyns et al. (2004) estimated that one infected animal will infect on average one susceptible
penmate during the nursery period. These results emphasize the importance of the
implementation of control strategies from an early age. In another transmission study, the same
authors (Meyns et al., 2006) showed that vaccination was efficacious to reduce clinical
symptoms and lung lesions, but that the transmission of M. hyopneumoniae was only
numerically lower in the vaccinated group (Rn= 2.38), compared to the non-vaccinated group
(Rn= 3.51). This implies that vaccination alone will not lead to elimination of this pathogen in a
herd. Whether these results are also valid under field conditions is not known.
1.1.7.3.3.
Development of new vaccines against M. hyopneumoniae
The first vaccines tested against M. hyopneumoniae were inactivated whole cell bacterins (Ross
et al., 1984; Kobisch et al., 1987) and adjuvanted bacterins are still the ones that are currently
36
CHAPTER 1: GENERAL INTRODUCTION
used commercially (Okada et al., 1999). Constant effort is being directed towards the
investigation of other M. hyopneumoniae strains, protective antigens, and routes of
administration that may offer a better protection against M. hyopneumoniae infections.
Apart from the conventional intramuscular administration, other alternative routes of
immunization have been described for pigs. These include intraperitoneal immunization
(Sheldrake et al., 1991; Ciprian et al., 1994), vaccines in aerosol (Murphy et al., 1993); oral
microcapsules (Lin et al., 2003); intradermal application (Jones et al., 2005) and intrapulmonic
immunization (Feng et al., 2010). Although most of these vaccines have shown to be efficacious
in the reduction of lung lesions and clinical symptoms under experimental and field conditions,
some of them have not been successful and have not demonstrated sufficient economic
improvement in performance parameters. More studies need to be conducted in order to validate
these vaccines under experimental and practical circumstances.
In addition, the route of vaccination can influence the type of immune response triggered in an
animal. For instance, the same subunit vaccine against M. hyopneumoniae (rLTBR1), composed
of R1 repeat region of P97 adhesin, induced a better Th2 response when administered
intramuscularly and induced a more accentuated Th1 response when administered intranasally in
mice (Conceição et al., 2006). Furthermore, some of these experimental vaccines require a more
labour intensive way of administration and are more expensive, which not always makes them
commercially justifiable (Jakab et al., 1995).
Aerosol and oral vaccine delivery have received special attention as alternative routes, since
these would allow mass vaccination, significantly reduce labour cost, increase mucosal immunity
in the respiratory tract (Lin et al., 2003) and decrease stress caused by individual handling during
37
CHAPTER 1: GENERAL INTRODUCTION
vaccination. Unfortunately, so far, they are less protective than the intramuscular vaccination
(Murphy et al., 1993).
Apart from the antigen in the vaccine, also the adjuvant plays a role in protection. Most
commercial formulations against M. hyopneumoniae contain oil-based adjuvants to prolong the
release of the antigen and stimulate the immune response of the host (Groth et al., 2001).
Nevertheless, the advantages of different adjuvants are still under debate. More studies are
needed to investigate the effect of different formulations.
Another concern in the search for new vaccines is that protective species-specific antigens
against M. hyopneumoniae still remain largely undefined and most of the formulations
encompassing single antigens tend to offer only partial protection. Recently, efforts are being
made on a molecular basis to map more epitopes that could potentially serve as components in
future vaccines (Talaat et al., 2005; Chen et al., 2008). The expression library immunization
(ELI), for instance, is a novel technique to screen genomes in order to identify potential vaccine
candidates (Talaat et al., 2005). Given its small genome, ELI was first used in M. pneumoniae. A
small genome is a characteristic also shared by M. hyopneumoniae. Up to date, three different
strains have been sequenced, namely J, 232 and 7448 (Minion et al., 2004; Vasconcelos et al.,
2005), making this pathogen a particularly suitable candidate for the ELI technique. The
utilization of ELI in a pig model and the development of improved vectors for screening
recombinant proteins were first reported by Moore et al. (2001). A recent study describes the
cloning, expression and purification of thirty new recombinant M. hyopneumoniae proteins using
E. coli as expression vector (Simionatto et al., 2010).
A combination of both novel and conventional methodologies has led to the identification of a
few specific antigens of M. hyopneumoniae, among which figures the lipoprotein P65 (Kim et
38
CHAPTER 1: GENERAL INTRODUCTION
al., 1990), cytosolic P36 protein, a L-lactate dehydrogenase (Strasser et al., 1991), surface
lipoproteins P46 (Futo et al., 1995), P97 protein (Zhang et al., 1995), P97R1, ribonucleotide
reductase – NrdF (Fagan et al., 1996) and lipoproteins Mhp378 and Mhp651 (Meens et al.,
2006). Although most of these molecules have a great potential as vaccine candidates, P97,
P97R1 and NrdF have been predominantly evaluated in various experimental studies that include
animal models (King et al., 1997; Fagan et al., 1997; Chen et al., 2001; 2003; 2006).
P97 plays an important role in the adherence of M. hyopneumoniae to the cilia (Zhang et al.,
1995; Minion et al., 2000) and ever since it was identified, it has been the most studied and best
defined potential protective antigen against M. hyopneumoniae. It has been used in experimental
subunit vaccines for mice and pig infection models (Chen et al., 2001; Conceicao et al., 2006).
The first use of P97 as a subunit vaccine provided only minimal, but not significative protection
against lung lesion (King et al., 1997), but subsequent studies have reported better protection and
higher antibody titers than with conventional vaccines (Chen et al., 2001). In another trial, an
attenuated Erysipelothrix rhusiopathiae YS-19 vaccine expressing the C-terminal portion of the
P97 adhesin was intranasally administered to pigs (Shimoji et al., 2003). It significantly reduced
the severity of pneumonic lung lesions, but failed to produce P97-specific serum antibodies.
Ogawa et al. (2009) reported that oral immunization with the same vaccine did not provide
successful results. The failure via the oral route could have been due to over-attenuation of the
strain. As a consequence, a live vaccine was developed against M. hyopneumoniae, using the E.
rhusiopathiae strain as a vector expressing the C-terminal domain of the P97 adhesin (Ogawa et
al., 2009). This time, the oral vaccine successfully induced protection against mycoplasmal
pneumonia, significantly reducing the severity of lung lesions.
39
CHAPTER 1: GENERAL INTRODUCTION
P97R1 (also called R1) is a repeat of P97 that contains both cilium and antibody binding sites
(Hsu and Minion, 1998; Hsu et al., 1997) and has an immunogenic effect independently from
other P97 regions (Minion et al., 2000). Generating anti-P97R1 mucosal immune response might
play a role in the prevention of colonization of the pig‘s respiratory tract by M. hyopneumoniae
(Sheldrake et al., 1993; Thacker et al., 2000). In an experiment performed by Chen et al. (2006),
mice immunized with the vaccine carrier Salmonella Typhimurium aroA CS332 generated a cellmediated response and serum IgG, but not mucosal IgA against P97R1, questioning the
suitability of the S. Typhimurium antigen-carrier as a vector encoding P97R1.
On the other hand, NrdF shows homology to the prokaryotic R2 subunit of ribonucleotide
reductase (Fagan et al., 1996). In an experiment, pigs immunized with a 11kDa protein
containing the C-terminal of NrdF, had significantly lower lung lesions after being infected with
M. hyopneumoniae, but there was no difference in weight gain in comparison with the control
animals. One year later, the same authors immunized mice orally with Salmonella Typhimurium
aroA strain SL3261 expressing NrdF fragment, producing significant specific IgA anti-NrdF in
the lungs, but there were no significant levels of IgG, IgM or IgA in the serum (Fagan et al.,
1997). A similar trial was performed in pigs, immunizing them orally with the same attenuated S.
Typhimurium aroA expressing a recombinant antigen of M. hyopneumoniae (NrdF) (Fagan et
al., 2001), demonstrating this time reduced lung lesions and higher daily weight gain compared
to the non vaccinated animals. An orally delivered vaccine in mice evoked NrdF-specific IFN-γ
and cellular mediated immunity by using a eukaryotic expression vector (Chen et al., 2006).
40
CHAPTER 1: GENERAL INTRODUCTION
Despite the immunizing properties of all of these proteins and the reduced severity of lung
lesions, none of them is currently able to offer total protection or a similar protection as the
commercial vaccines. Multivalent formulations could be another alternative in offering a better
protection against M. hyopneumoniae (Chen et al., 2008). Inoculation with plasmid DNA,
encoding immunogenic proteins for a specific pathogen, has emerged as a novel approach for
developing new generation vaccines (Dhama et al., 2008). Cocktail DNA vaccines elicit both cell
mediated and humoral immunity. They can be administered in different ways (intramuscular,
subcutaneous or oral), and they can also contain recombinant plasmids with multiple potential
antigens. A multivalent DNA vaccine with different immunogenic proteins against M.
hyopneumoniae (P36, P46, NrdF, and P97or P97R1) has been tested in mice (Chen et al., 2008).
The major findings were that intramuscular immunization induced a strong Th1 immune
response, whereas the humoral response was elicited only by P46. Subcutaneous immunization,
on the other hand, triggered both humoral and Th1 response. In addition, P97 was not recognized
by serum antibodies in mice immunized with a commercial bacterin, which might be an
indication of the lack of expression of this antigen in inactivated whole-cell vaccines.
The major limitations faced nowadays with DNA vaccines is that while they enable a long
lasting protection effect in small experimental animals, less potent immune responses are
achieved in larger animals. Moreover, plasmid DNA is easily degradable in vivo, requiring better
delivery systems for the vaccine to remain efficacious (Dhama et al., 2008).
Although the novel methodologies and new antigens may show promising effects for the control
of M. hyopneumoniae infections, many hurdles are still to be taken to achieve a better control
than with the current commercially available vaccines.
41
CHAPTER 1: GENERAL INTRODUCTION
References
Amass, S.F., Clark, L.K. 1999. Biosecurity considerations for pork production units. Swine
Health and Prod. 7, 217-228.
Amass, S.F., Stevenson, G.W., Anderson, C., Grote, L.A., Dowell, A., Vyverberg, B.D., Kanitz,
C., Ragland, D., 2000. Investigation of people as mechanical vectors for porcine reproductive
and respiratory syndrome virus. Swine Health Prod. 8, 161–166.
Asai, T., Okada, M., Ono, M., Mori, Y., Yokomizo, Y., Sato, S., 1994. Detection of interleukin-6
and prostaglandin E2 in bronchoalveolar lavage fluids of pigs experimentally infected with
Mycoplasma hyponeumoniae. Vet. Immunol. Immunopathol. 44, 97-102.
Asai, T., Okada, M., Yokomizo, Y., Sato, S. and Mori, Y., 1996. Suppressive effect of
bronchoalveolar lavage fluid from pigs infected with Mycoplasma hyopneumoniae on
chemiluminescence of porcine peripheral neutrophils. Vet. Immunol. Immunopathol. 51, 325–
331.
Baarsch, M.J., Scamurra, R.W., Burger, K., Foss, D.L., Maheswaran, S.K., Murtaugh, M.P.,
1995. Inflammatory cytokine expression in swine experimentally infected with Actinobacillus
pleuropneumoniae. Infect. Immun. 63, 3587-3594.
Baccaro, M.R., Hirose, F., Umehara, O., Goncalves, L.C., Doto, D.S., Paixao, R., Shinya, L.T.,
Moreno, A.M., 2006. Comparative efficacy of two single-dose bacterins in the control of
Mycoplasma hyopneumoniae in swine raised under commercial conditions in Brazil. Vet. J. 172,
526-531.
Bandrick, M., Pieters, M., Pijoan, C., Molitor, T.W., 2008. Passive transfer of maternal
Mycoplasma hyopneumoniae-specific cellular immunity to piglets. Clin. Vaccine Immunol. 15,
540-543.
Leeanne E. Bargen, L. E., 2004. A system response to an outbreak of enzootic pneumonia in
grow/finish pigs. Can Vet J. 45, 856–859.
42
CHAPTER 1: GENERAL INTRODUCTION
Bargen, L., 2004. A system response to an outbreak of enzootic pneumonia in grow/finish pigs.
Can Vet J. 45, 856–859.
Baskerville, A., 1972. Development of the early lesions in experimental enzootic pneumonia of
pigs: an ultrastructural and histological study. Res. Vet. Sci. 13, 570-578.
Batista, L., Pijoan, C., Ruiz, A., Utrera, V., and Dee, S., 2004. Assessment of transmission of
Mycoplasma hyopneumoniae by personnel, J. Swine Health Prod. 12, 75–77.
Blanchard, B., Vena, M.M., Cavalier, A., Le, L.J., Gouranton, J., Kobisch, M., 1992. Electron
microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma
hyopneumoniae. Vet. Microbiol. 30 , 329-341.
Bochev, 2008. Porcine Respiratory Disease Complex- a review. Bulg. J. Vet. Med. 11, 219-234
Bousquet, E., Pommier, P., Wessel-Robert, S., Morvan, H., Benoit-Valiergue, H., Laval, A.,
1998. Efficacy of doxycycline in feed for the control of pneumonia caused by Pasteurella
multocida and Mycoplasma hyopneumoniae in fattening pigs. Vet. Rec. 143, 269-272.
Bradbury, J., 1998. Identification of Mycoplasmas by Immunofluorescence. Mycoplasma
Protocols. Methods Molecular Biol. 104, 119-125.
Braun, V., Sieglin, U., 1970. The covalent murein-lipoprotein structure of the Escherichia coli
cell wall. The attachment site of the lipoprotein on the murein. Eur J Biochem. 13, 336-46.
Burgi, E., Seiz, O.,
Bertschinger, H. U., 1990. Lack of Transmission of Mycoplasma
hyopneumoniae from Dam to Fetus in Experimentally Infected Pregnant Gilts. In: Proc. 11th
Congr., IPVS, Lausanne, Switzerland, p. 89.
43
CHAPTER 1: GENERAL INTRODUCTION
Butera, M., Smith, J. H. , Morrison, W. D. , Hacker, R. R., Kains, F. A., and Ogilvie, J. R., 1991.
Concentration of respirable dust and bioaerosols and identification of certain microbial types in a
hog-growing facility. Canadian Journal of Animal Science 71, 271-277.
Calsamiglia, M., Pijoan, C., Trigo, A., 1999a. Application of a nested polymerase chain reaction
assay to detect Mycoplasma hyopneumoniae from nasal swabs. J. Vet. Diagn. Invest 11, 246251.
Calsamiglia M., Pijoan, C., Bosch, G., 1999b. Profiling M. hyopneumoniae in famrs using
serology and a nested PCR techinique. Swine Health and Prod. 7, 263-268.
Calsamiglia, M., Collins, J.E., Pijoan, C., 2000a. Correlation between the presence of enzootic
pneumonia lesions and detection of Mycoplasma hyopneumoniae in bronchial swabs by PCR.
Vet. Microbiol. 76, 299-303.
Calsamiglia, M., Pijoan, C., 2000b. Colonisation state and colostral immunity to Mycoplasma
hyopneumoniae of different parity sows. Vet. Rec. 146, 530-532.
Calus, D., Baele, M., Meyns, T., de, K.A., Butaye, P., Decostere, A., Haesebrouck, F., Maes, D.,
2007. Protein variability among Mycoplasma hyopneumoniae isolates. Vet. Microbiol. 120, 284291.
Calus, D., Maes, D., Meyns, T., Pasmans, F., Haesebrouck, F., 2009. In vivo virulence of
Mycoplasma hyopneumoniae isolates does not correlate with in vitro adhesion assessed by a
microtitre plate adherence assay. J. Appl. Microbiol. 106, 1951-1956.
Calus, D., 2010a. Phenotypic characterization of Mycoplasma hyopneumoniae isolates of known
virulence. Faculty of Veterinary Medicine, Thesis pp. 14-39.
Calus, D., Maes, D., Vranckx, K., Villareal, I., Pasmans, F., Haesebrouck, F., 2010b. Validation
of ATP luminometry for rapid and accurate titration of Mycoplasma hyopneumoniae in Friis
medium and a comparison with the color changing units assay. J Microbiol Methods. Article in
press.
44
CHAPTER 1: GENERAL INTRODUCTION
Cardona, A.C., Pijoan, C., Dee, S.A., 2005. Assessing Mycoplasma hyopneumoniae aerosol
movement at several distances. Vet. Rec.. 156, 91-92.
Caron, J., Ouardani, M., Dea, S., 2000. Diagnosis and differentiation of Mycoplasma
hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36
and p46 genes. J. Clin. Microbiol. 38, 1390-1396.
Caruso, J.P., Ross, R.F., 1990. Effects of Mycoplasma hyopneumoniae and Actinobacillus
(Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine. Am. J.
Vet. Res. 51, 227-231.
Chambaud, I., Wróblewski, H., Blanchard, A., 1999. Interactions between mycoplasma
lipoproteins and the host immune system. Trends in Microbiology 7, 493-499.
Chen, J.R., Liao, C.W., Mao, S.J., Weng, C.N., 2001. A recombinant chimera composed of
repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo
evaluation of specific IgG response in mice and pigs. Vet. Microbiol. 80, 347-357.
Chen, Y.L., Wang, S.N., Yang, W.J., Chen, Y.J., Lin, H.H., Shiuan, D., 2003. Expression and
immunogenicity of Mycoplasma hyopneumoniae heat shock protein antigen P42 by DNA
vaccination. Infect. Immun. 71, 1155-1160.
Chen, A.Y., Fry, S.R., Forbes-Faulkner, J, Mukkur, T.K., 2006a. Comparative immunogenicity
of M. hyopneumoniae NrdF encoded in different expression systems delivered orally via
attenuated S. typhimurium aroA in mice. Vet. Microbiol.. 31, 252-259.
Chen, A.Y., Fry, S.R., Forbes-Faulkner, J., Daggard, G., Mukkur, T.K., 2006b. Evaluation of the
immunogenicity of the P97R1 adhesin of Mycoplasma hyopneumoniae as a mucosal vaccine in
mice. J. Med. Microbiol. 55, 923-929.
Chen, A.Y., Fry, S.R., Daggard, G.E., Mukkur, T.K., 2008. Evaluation of immune response to
recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail
DNA and/or recombinant protein vaccines in mice. Vaccine 26, 4372-4378.
45
CHAPTER 1: GENERAL INTRODUCTION
Choi, C., Kwon, D., Jung, K., Ha, Y., Lee, Y.H., Kim, O., Park, H.K., Kim, S.H., Hwang, K.K.,
Chae, C., 2006. Expression of inflammatory cytokines in pigs experimentally infected with
Mycoplasma hyopneumoniae. J. Comp Pathol. 134, 40-46.
Ciprian, A., Cruz, T.A., de la Garza, M., 1994. Mycoplasma hyopneumoniae: interaction with
other agents in pigs, and evaluation of immunogens. Arch. Med. Res. 25, 235-239.
Ciprian, A., Pijoan, C., Cruz, T., Camacho, J., Tortora, J., Colmenares, G., Lopez-Revilla, R., de
la Garza, M., 1988. Mycoplasma hyopneumoniae increases the susceptibility of pigs to
experimental Pasteurella multocida pneumonia. Can. J. Vet. Res. 52, 434-438.
Clark, L., Freeman, M., Scheidt, A., Knox K., 1991. Investigating the transmisson of
Mycoplasma hyopneumoniae in a swine herd with enzootic pneumonia. Vet. Med. 86, 543-550.
Conceicao, F.R., Moreira, A.N., Dellagostin, O.A., 2006. A recombinant chimera composed of
R1 repeat region of Mycoplasma hyopneumoniae P97 adhesin with Escherichia coli heat-labile
enterotoxin B subunit elicits immune response in mice. Vaccine 24, 5734-5743.
DeBey, M.C., Jacobson, C.D., Ross, R.F., 1992. Histochemical and morphologic changes of
porcine airway epithelial cells in response to infection with Mycoplasma hyopneumoniae. Am. J.
Vet. Res. 53, 1705-1710.
DeBey, M.C., Roth, J.A., Ross, R.F., 1993. Enhancement of the increase in intracellular calcium
concentration in stimulated neutrophils by Mycoplasma hyopneumoniae. Vet. Res. Commun. 17,
249-257.
DeBey, M.C., Ross, R.F., 1994. Ciliostasis and loss of cilia induced by Mycoplasma
hyopneumoniae in porcine tracheal organ cultures. Infect. Immun. 62, 5312-5318.
Dee, S., 1994. Apparent prevention of Mycoplasma hyopneumoniae infection in growing pigs
with a low-cost modified medicated-early-weaning program. Swine Health and Prod. 2, 7-12.
Dee, S., Otake, S., Oliveira, S., Deen, J., 2009. Evidence of long distance airborne transport of
porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Vet. Res.
40, 39.
46
CHAPTER 1: GENERAL INTRODUCTION
Dhama, K., Mahendran, M, Gupta, P. K., Rai, A., 2008. DNA vaccines and their applications in
veterinary practice: current perspectives. Vet. Res. Commun. 32, 341-356.
Diekman, M.A., Scheidt, A.B., Grant, A.L. , Kelly, D.T. , Sutton, A.L. , Martin, T.G. , and
Cline, T.R. , 1999. Effect of vaccination against Mycoplasma hyopneumoniae on health, growth,
and pubertal status of gilts exposed to moderate ammonia concentrations in all-in-all-out versus
continuous-flow system, swine health. Swine Health Prod. 7, 55–61.
Dohoo, I., Martin, W., Stryhn, H., 2009. Veterinary Epidemiologic Research. In: Concepts of
infectious disease epidemiology. Canada, Friesens Manitoba p. 715-725.
Done, S.. 1991. Pneumonia, some aspects of treatment and control. Pig Vet. J. 27, 30-49.
Done, S., 1996. Enzootic pneumonia (mycoplasmosis) revisited. Pig J. 38, 40-61.
Erlandson, K.R. , Thacker, B.J. , Bush E.J. , and Thacker, E.L., 2002.
Mycoplasma
hyopneumoniae seroprevalence and control strategies on farms participating in the national
animal health monitoring system (NAHMS) swine 2000 survey, Proc. 33th Annu. Meet. Am.
Assoc. Swine Pract. Kansas City, Missouri, 31–33.
Fagan, P.K., Djordjevic, S.P., Eamens, G.J., Chin, J., Walker, M.J., 1996. Molecular
characterization of a ribonucleotide reductase (nrdF) gene fragment of Mycoplasma
hyopneumoniae and assessment of the recombinant product as an experimental vaccine for
enzootic pneumonia. Infect. Immun. 64, 1060-1064.
Fagan, P.K., Djordjevic, S.P., Chin, J., Eamens, G.J., Walker, M.J., 1997. Oral immunization of
mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma
hyopneumoniae antigen (NrdF). Infect. Immun. 65, 2502-2507.
Fagan, P.K., Walker, M.J., Chin, J., Eamens, G.J., Djordjevic, S.P., 2001. Oral immunization of
swine with attenuated Salmonella typhimurium aroA SL3261 expressing a recombinant antigen
47
CHAPTER 1: GENERAL INTRODUCTION
of Mycoplasma hyopneumoniae (NrdF) primes the immune system for a NrdF specific secretory
IgA response in the lungs. Microb. Pathog. 30, 101-110.
Fano, E., Pijoan, C., Dee, S., 2005a. Dynamics and persistence of Mycoplasma hyopneumoniae
infection in pigs. Can. J. Vet. Res. 69, 223-228.
Fano, E., Pijoan, C., Dee, S., 2005b. Evaluation of the aerosol transmission of a mixed infection
of Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus. Vet.
Rec. 157, 105-108.
Fano, E., Pijoan, C., Dee, S., Torremorell, M., 2006. Assessment of the effect of sow parity on
the prevalence of Mycoplasma hyopneumoniae in piglets at weaning. In: Proceedings of the 19th
IPVS congress, Copenhagen, Denmark, Vol. I, 96.
Fano, E., Pijoan, C., Dee, S., 2007a. Infection dynamics of porcine reproductive and respiratory
syndrome virus in a continuous-flow population of pigs also infected with Mycoplasma
hyopneumoniae. Vet. Rec. 161, 515-520.
Fano, E., Pijoan, C., Dee, S., Deen, J., 2007b. Effect of Mycoplasma hyopneumoniae
colonization at weaning on disease severity in growing pigs. Can. J. Vet. Res. 71, 195-200.
Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F., Meyling, A., 1992. A monoclonal blocking ELISA
detecting serum antibodies to Mycoplasma hyopneumoniae. Vet. Microbiol. 30, 35-46.
Feng, ZX, Shao GQ, Liu, MJ, Wang HY, Gan Y, Wu XS., 2010. Development and validation of
a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection. Vet. Microbiol.. 14,
410-416.
Ferreira, H.B., 2007. A preliminary survey of M. hyopneumoniae virulence factors based on
comparative genomic analysis. Genet. Mol. 30, 245-255.
Flesja, K. and Ulvesaeter, H., 1980. Pathological lesions in swine at slaughter. Acta Vet. Scand.,
74: 1-22.
48
CHAPTER 1: GENERAL INTRODUCTION
Friis, N.F., 1974. Mycoplasmas in pigs with special regard to the respiratory tract. Thesis. DSR
Forlag. Royal Veterinary and Agricultural University, Copenhagen.
Furlong, S.L., Turner, A.J., 1975. Isolation of Mycoplasma hyopneumoniae and its association
with pneumonia of pigs in Australia. Aust. Vet. J. 51, 28-31.
Futo, S., Seto, Y., Mitsuse, S., Mori, Y., Suzuki, T., Kawai, K., 1995. Molecular cloning of a 46kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of
CGG codon usage for arginine. J. Bacteriol. 177, 1915-1917.
Gebruers, F., Calus, D., Maes, D., Villarreal, I., Pasmans, F., Haesebrouck, F., 2008. A set of
four nested PCRs to detect different strains of Mycoplasma hyopneumoniae in biological
samples. In: Proceedings of the 17th Congress of the International Organisation for Mycoplasmology, Tianjin, China, pp. 90.
Geers, R., Dellaert, B., Goedseels, V., Hoogerbrugge, A., Vranken, E., Maes, F. and Berckmans,
D., 1989. An assessment of optimal air temperatures in pig houses by the quantification of
behavioural and health-related problems. Anim. Pmd. 48, 571-578.
Georgakis, A.D., Bourtzi-Hatzopoulou, E., Kritas, S.K., Balkamos, G.C., Kyriakis, S.C ., 2002.
A study on the Porcine Respiratory Disease Syndrome (PRDC): Update review and proposed
measures for its control. J Hellenic Vet Med Society 53, 265-271.
Goodwin, R.F., 1965. The phenomenon of suppressed respiratory disease in the litters of older
sows. Vet. Rec. 77, 383-387.
Goodwin, R. F. W. 1972: The survival of Mycoplasma suipneumoniae in liquid medium, on
solid medium and in pneumonic tissue. Res. Vet. Sci. 13, 203-204.
Goodwin, R.F., 1985a. Apparent reinfection of enzootic-pneumonia-free pig herds: search for
possible causes. Vet. Rec. 116, 690-694.
49
CHAPTER 1: GENERAL INTRODUCTION
Goodwin, R.F., 1985b. In vitro activity of tiamulin against porcine mycoplasmas. Res. Vet. Sci.
38, 124-125.
Gordon, W., 1963. Environmental studies in pig housing. The effects of housing on the degree
and incidence of pneumonia in bacon pigs. Br. Vet. J., 119, 307-314.
grosse Beilage, E., Schreiber, A., 2005. Impfung von Sauen gegen Mycoplasma hyopneumoniae
mit Hyoresp®/ Vaccination of sows against Mycoplasma hyopneumoniae with Hyoresp®.
Deutsche tierärztliche Wochenschrift 112, 256-261.
grosse Beilage, E., Rohde, N., Krieter, J., 2009. Seroprevalence and risk factors associated with
seropositivity in sows from 67 herds in north-west Germany infected with Mycoplasma
hyopneumoniae. Prev. Vet. Med. 88, 255-263.
Groth, D., Rapp-Gabrielson, V. , 2001. Evaluation of M+PAC in one and two dose-regimens
against competitor one-dose Mycoplasma hyopneumoniae bacterins. Proceedings of the Allan D.
Leman Swine Conference, Recent Research Reports, Supplement, University of Minnesota,
USA, 28, 41.
Haesebrouck, F., Pasmans, F., Chiers, K., Maes, D., Ducatelle, R., Decostere, A., 2004. Efficacy
of vaccines against bacterial diseases in swine: what can we expect? Vet. Microbiol. 100, 255268.
Hannan, P.C., Bhogal, B.S., Fish, J.P., 1982. Tylosin tartrate and tiamutilin effects on
experimental piglet pneumonia induced with pneumonic pig lung homogenate containing
mycoplasmas, bacteria and viruses. Res. Vet. Sci. 33, 76-88.
Hannan, P.C., Windsor, H.M., Ripley, P.H., 1997. In vitro susceptibilities of recent field isolates
of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae to valnemulin (Econor), tiamulin
and enrofloxacin and the in vitro development of resistance to certain antimicrobial agents in
Mycoplasma hyopneumoniae. Res. Vet. Sci. 63, 157-160.
50
CHAPTER 1: GENERAL INTRODUCTION
Harasawa, R., Koshimizu, K., Takeda, O., Uemori, T., Asada, K., Kato, I., 1991. Detection of
Mycoplasma hyopneumoniae DNA by the polymerase chain reaction. Mol. Cell Probes 5, 103109.
Harris, L.D., Alexander, T.J., 1999. Methods of disease control. In: Straw B, D‘Allaire S,
Mengeline W, Taylor D, eds. Diseases of Swine, 8th ed. Ames, Iowa: Iowa State Univ Pr,
pp.1077–1078.
Heber, A. J., M. Stroik, J. M. Faubion, and L. H. Willard. 1988. Size distribution and
identification of aerial dust particles in swine finishing building. Transactions of the ASAE 31,
882-887.
Hege, R., Zimmermann, W., Scheidegger, R., Stark, K.D., 2002. Incidence of reinfections with
Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae in pig farms located in
respiratory-disease-free regions of Switzerland--identification and quantification of risk factors.
Acta Vet. Scand. 43, 145-156.
Heinonen, M., Gröhn, Y., Saloniemi, H., Eskola, E., Tuovinen, V., 2001. The effects of health
classification and housing and management of feeder pigs on performance and meat inspection
findings of all-in–all-out swine-finishing herds. Prev. Vet. Med. 49, 41-54.
Henderson, B., Poole, S., Wilson, M., 1996. Bacterial modulins: a novel class of virulence
factors which cause host tissue pathology by inducing cytokine synthesis. Microbiological
reviews 60, 316-341.
Hirst, J. M., 1995. Bioaersols: Introduction, retrospect and prospect. Bioaerosol Handbook. Boca
Raton, Florida: CRC Lewis Publishers p. 5-14.
Hsu, T., Artiushin, S., Minion, F.C., 1997. Cloning and functional analysis of the P97 swine
cilium adhesin gene of Mycoplasma hyopneumoniae. J. Bacteriol. 179, 1317-1323.
51
CHAPTER 1: GENERAL INTRODUCTION
Hsu, T., Minion, F.C., 1998. Identification of the cilium binding epitope of the Mycoplasma
hyopneumoniae P97 adhesin. Infect. Immun. 66, 4762-4766.
Hurnik, J. F. 1992. Animal responses to the environment. In: C. Philips and D. Piggins (Ed.)
Behaviour in Farm Animals and Their Environment. C.A.B. International Publishers,
Wallingford, U.K.
Hurnik, D., Hanna, P.E., Dohoo, I.R., 1993. Evaluation of rapid gross visual appresial of swine
lungs at slaughter as a diagnostic screen for enzootic pneumonia. Can. J. Vet. Res. 57, 37-41.
Hurnik, D., Dohoo, I.R, Bate, L.A., 1994a. Types of farm management as risk factors for swine
respiratory disease. Prev. Vet. Red. 20, 147-157.
Hurnik, D., Dohoo, I.R, Donald, A., Robinson, P, 1994b. Factor analysis of swine farm
management practices on Prince Edward Island. Prev. Vet. Red. 20, 135-146.
Ice, A. D., Grant, A. L., Clark, L.K., Cline, T. R., Einstein, M. E., and Diekman, M. A., 1997.
Health and lean growth performance of barrows reared in all-in, all-out or continuous flow
facilities with or without antibiotic. Purdue University Swine Day 78-81.
Inamoto, T., Takahashi, H., Yamamoto, K., Nakai, Y., Ogimoto, K., 1994. Antibiotic
susceptibility of Mycoplasma hyopneumoniae isolated from swine. J. Vet. Med. Sci. 56, 393394.
Jacques, M., Blanchard, B., Foiry, B., Girard, C., Kobisch, M., 1992. In vitro colonization of
porcine trachea by Mycoplasma hyopneumoniae. Ann. Rech. Vet. 23, 239-247.
Jakab, L., Rafai, P., Szabo, I., 1995. Study of the efficacy of active immunization against
Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae on a large-scale pig farm.
Proceedings of the Symposium of the International Society for Animal Hygiene p. 676-677.
52
CHAPTER 1: GENERAL INTRODUCTION
Jenkins, C., Wilton, J.L., Minion, F.C., Falconer, L., Walker, M.J., Djordjevic, S.P., 2006. Two
domains within the Mycoplasma hyopneumoniae cilium adhesin bind heparin. Infect. Immun. 74,
481-487.
Jensen, D., Ersboll, A., and Nielsen, J. , 2002. A meta-analysis comparing the effect of vaccines
against Mycoplasma hyopneumoniae on daily weight gain in pigs, Prev. Vet. Med. 54, 265–278.
Jirawattanapong, P., Stockhofe-Zurwieden, N., van Leengoed, L., Wisselink, H., Raymakers, R.,
Cruijsen, T., van der Peet-Schwering, C., Nielen, M., van Nes, A., 2010. Pleuritis in slaughter
pigs: relations between lung lesions and bacteriology in 10 herds with high pleuritis. Res. Vet.
Sci. 88, 11-5.
Jolie, R., Bäckström, L., Pinckney, R., 1998. Ascarid infection and respiratory health in feeder
pigs raised on pasture or in confinement. J. Swine Health Prod. 6, 115–120.
Jones, G., Rapp-Gabrielson, V., Wilke, R., Thacker, E., Thacker, B., Gergen, L., Sweeney, D.,
Wasmoen, T., 2005. Intradermal vaccination for Mycoplasma hyopneumoniae. J. Swine Health
Prod. 13, 19-27.
Jorsal, S. and Thomsen, B., 1988. A cox regression analysis of risk factors related to
Mycoplasma suipneumoniae reinfection in Danish SPF-herds. Acta Vet. Scand. [Suppl.], 84,
436-438.
Jouglar, J., Borne, P., Sionneau, G., Bardon, T. & Eclache, D. (1993). Evaluation de l‘intérêt de
l‘association tiamuline-oxytétracycline dans la prévention des bronchopneumonies du porc
charcutier. Revue de Médécine Vétérinaire 144, 981-988.
Kim, M.F., Heidari, M.B., Stull, S.J., McIntosh, M.A., Wise, K.S., 1990. Identification and
mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein
expressed in Escherichia coli from a cloned genomic fragment. Infect. Immun. 58, 2637-2643.
53
CHAPTER 1: GENERAL INTRODUCTION
Kobisch, M., Quillien, L., Tillon, J.P., Wroblewski, H., 1987. The Mycoplasma hyopneumoniae
plasma membrane as a vaccine against porcine enzootic pneumonia. Ann. Inst. Pasteur Immunol.
138, 693-705.
Kobisch, M., Blanchard, B., Portier, M. and Le Potier, M., 1993. Mycoplasma hyopneumoniae
infection in pigs: duration of the disease and resistance to reinfection. Vet. Res., 24: 67-77.
Kobisch, M., Friis, N. F., 1996. Swine mycoplasmoses. Rev. Sci. Tech.15:1569–1605.
King, K.W., Faulds, D.H., Rosey, E.L., Yancey, R.J., Jr., 1997. Characterization of the gene
encoding Mhp1 from Mycoplasma hyopneumoniae and examination of Mhp1's vaccine potential.
Vaccine 15, 25-35.
Kurkjian, K., Little, S., 2003. Routes of transmission of infectious diseases agents. Modes of
introduction of exotic animal disease agents.The University of Georgia College of Veterinary
Medicine. The University of Georgia College of Veterinary Medicine.
Kurth, K.T., Hsu, T., Snook, E.R., Thacker, E.L., Thacker, B.J., Minion, F.C., 2002. Use of a
Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal
sampling sites in swine. J. Vet. Diagn. Invest 14, 463-469.
Kwon, D., Choi, C., and Chae, C., 2002. Chronologic localization of Mycoplasma
hyopneumoniae in experimentally infected pigs. Vet. Pathol. 39, 584–587.
Le Carrou, J., Laurentie, M., Kobisch, Gautier-Bouchardon, A.V., 2006. Persistence of
Mycoplasma hyopneumoniae in Experimentally Infected Pigs after Marbofloxacin Treatment
and Detection of Mutations in the parC Gene. Antimic. Agents and Chemother. 50, 1959–1966.
Le Potier, M., Abiven, P., Kobisch, M., Crevat, D., and Desmettre, P., 1994. A blocking ELISA
using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae. Res.
Vet. Sci. 56, 338–345.
54
CHAPTER 1: GENERAL INTRODUCTION
Lebret, B., Meunier-Salaun, C., Foury, A., Mormede P., Dransfield, E., Dourmand, J. Y., 2006.
Influence of rearing conditions on performance, behavioral, and physiological responses of pigs
to preslaughter handling, carcass traits, and meat quality. J Anim. Sci. 84, 2436-2447.
Leneveu, P., Robert, N., Keïta, A., Pagot, E., Pommier P., and Tessier, P., 2005. Lung lesions in
pigs at slaughter: a 2-year epidemiological study in France. Int. J. Appl. Res. Vet. Med. 3, 259–
265.
Leon, E., Madec, F., Taylor N., Kobisch, M., 2001. Seroepidemiology of Mycoplasma
hyopneumoniae in pigs from farrow-to-finish farms. Vet. Microbiol. 78, 331-341.
Li, Y.Z., Ho, Y.P., Chen, S.T., Chiou, T.W., Li, Z.S., Shiuan, D., 2009. Proteomic comparative
analysis of pathogenic strain 232 and avirulent strain J of Mycoplasma hyopneumoniae.
Biochemistry (Mosc.) 74, 215-220.
Lin, J.H., Weng, C.N., Liao, C.W., Yeh, K.S., Pan, M.J., 2003. Protective effects of oral
microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method. J.
Vet. Med. Sci. 65, 69-74.
Lium, B.M., Falk, K., 1991. An abattoir survey of pneumonia and pleuritis in slaughter weight
swine from 9 selected herds. Acta. Vet. Scand. 32, 55-65.
Lorenzo, H., Quesada, O., Assuncao, P., Castro, A., Rodriguez, F., 2006. Cytokine expression in
porcine lungs experimentally infected with Mycoplasma hyopneumoniae. Vet. Immunol.
Immunopathol. 109, 199-207.
Madec, F., Kobisch, M., 1982. Bilan lesionnel des poumons de porcs charcutiers a l‘abattoir.
Journ. Rech. Porc., 14, 405-412.
Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Lein, A., Vrijens, B., de, K.A.,
1998. The effect of vaccination against Mycoplasma hypopneumoniae in pig herds with a
continuous production system. Zentralbl. Veterinarmed. B 45, 495-505.
Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., de, K.A., 1999a. Risk
indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine influenza viruses and
55
CHAPTER 1: GENERAL INTRODUCTION
Aujeszky's disease virus in slaughter pigs from fattening pig herds. Zentralbl. Veterinarmed. B
46, 341-352.
Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., Verbeke, W., Viaene,
J., de, K.A., 1999b. Effect of vaccination against Mycoplasma hyopneumoniae in pig herds with
an all-in/all-out production system. Vaccine 17, 1024-1034.
Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., de, K.A., 2000. Herd
factors associated with the seroprevalences of four major respiratory pathogens in slaughter pigs
from farrow-to-finish pig herds. Vet. Res. 31, 313-327.
Maes, D., Verbeke, W., Vicca, J., Verdonck, M., and de Kruif, A., 2003. Benefit to cost of
vaccination against mycoplasma hyopneumoniae in pig herds under Belgian market conditions
from 1996 to 2000. Livestock Production Science 83, 85-93.
Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M., Haesebrouck, F., 2008. Control of
Mycoplasma hyopneumoniae infections in pigs. Vet. Microbiol. 126, 297-309.
Mare, C.J., Switzer, W.P., 1965. New Species: Mycoplasma hyopneumoniae: A causative agent
of virus pig pneumonia. Vet. Med. Small Anim Clin. 60, 841-846.
Martelli, P., Terreni, M., Guazzetti, S., Cavirani, S., 2006. Antibody response to Mycoplasma
hyopneumoniae infection in vaccinated pigs with or without maternal antibodies induced by sow
vaccination. J. Vet. Med. B Infect. Dis. Vet. Public Health 53, 229-233.
Martinez, J., Peris, B., Gomez, E.A., Corpa, J.M., 2009. The relationship between infectious and
non-infectious herd factors with pneumonia at slaughter and productive parameters in fattening
pigs. Vet. J. 179, 240-246.
Mateusen, B., Maes, D., Hoflack, G., Verdonck, M., de, K.A., 2001. A comparative study of the
preventive use of tilmicosin phosphate (Pulmotil premix) and Mycoplasma hyopneumoniae
vaccination in a pig herd with chronic respiratory disease. J. Vet. Med. B Infect. Dis. Vet. Public
Health 48, 733-741.
56
CHAPTER 1: GENERAL INTRODUCTION
Mateusen, B., Maes, D., Van, G.M., Verdonck, M., de, K.A., 2002. Effectiveness of treatment
with lincomycin hydrochloride and/or vaccination against Mycoplasma hyopneumoniae for
controlling chronic respiratory disease in a herd of pigs. Vet. Rec. 151, 135-140.
Mattsson, J. G., Bergstom, K., Wallgren, P., Johansson, E., 1995. Detection of Mycoplasma
hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S RNA gene.
J.Clin.Microbiol. 33, 893-897.
McKelvie, J., Morgan, J.H., Nanjiani, I.A., Sherington, J., Rowan, T.G., Sunderland, S.J., 2005.
Evaluation of tulathromycin for the treatment of pneumonia following experimental infection of
swine with Mycoplasma hyopneumoniae. Vet Ther. 6, 197-202.
Mayor, D., Zeeh, F., Frey, J., Kuhnert, P., 2007. Diversity of Mycoplasma hyopneumoniae in pig
farms revealed by direct molecular typing of clinical material. Vet. Res. 38, 391-398.
Messier, S., Ross, R.F., Paul, P.S., 1990. Humoral and cellular immune responses of pigs
inoculated with Mycoplasma hyopneumoniae. Am. J. Vet. Res. 51, 52-58.
Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F., de, K.A., 2004. Quantification of
the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments. Prev.
Vet. Med. 66, 265-275.
Meyns, T., Dewulf, J., de, K.A., Calus, D., Haesebrouck, F., Maes, D., 2006. Comparison of
transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations.
Vaccine 24, 7081-7086.
Meyns, T., Maes, D., Calus, D., Ribbens, S., Dewulf, J., Chiers, K., de, K.A., Cox, E., Decostere,
A., Haesebrouck, F., 2007. Interactions of highly and low virulent Mycoplasma hyopneumoniae
isolates with the respiratory tract of pigs. Vet. Microbiol. 120, 87-95.
Miles, R.J., Taylor R.R., Varsani, H., 1991. Oxygen uptake and H2O2 production by fermentative
Mycoplasma spp., J. Med. Microbiol. 34, 219–223.
57
CHAPTER 1: GENERAL INTRODUCTION
Minion, F.C., Adams, C., Hsu, T., 2000. R1 region of P97 mediates adherence of Mycoplasma
hyopneumoniae to swine cilia. Infect. Immun. 68, 3056-3060.
Minion, F.C., Lefkowitz, E.J., Madsen, M.L., Cleary, B.J., Swartzell, S.M., Mahairas, G.G.,
2004. The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine
mycoplasmosis. J. Bacteriol. 186, 7123-7133.
Moore, R., Lenghaus, C., Sheedy, S., Doran, T., 2001. Improved vectors for expression library
immunization application to Mycoplasma hyopneumoniae infection in pigs. Vaccine 20, 115120.
Moorkamp, L., Hewicker-Trautwein, M., grosse Beilage, E., 2009. Occurrence of Mycoplasma
hyopneumoniae in coughing piglets (3-6 weeks of age) from 50 herds with a history of endemic
respiratory disease. Transbound. Emerg. Dis. 56, 54-56.
Moreau, I.A., Miller, G.Y., Bahnson, P.B., 2004. Effects of Mycoplasma hyopneumoniae vaccine
on pigs naturally infected with M. hyopneumoniae and porcine reproductive and respiratory
syndrome virus. Vaccine 22, 2328-2333.
Morris, C., Gardner, I., Hietala, S., Carpenter, T., Anderson, R. and Parker, K.,1994. Persistence
of passively acquired antibodies to Mycoplasma hyopneumoniae in a swine herd. Prev. Vet.
Med. 21, 29-41.
Morris, C., Gardner, I., Hietala, S. and Carpenter, T., 1995. Enzootic pneumonia: comparison of
cough and lung lesions as predictors of weight gain in swine. Can J. Vet. Res. 59, 197-204.
Morris, P.J., Morris, M., Sanford, S.E., 2001. Comparison of performance parameters of pigs
vaccinated with Ingelvac M. hyo 1-dose bacterin vs Respisure Mycoplasma hyopneumoniae
bacterin. In: Proceedings of the 32nd Annual Meeting of the American Association of Swine
Veterinarians, Nashville, Tennessee, 157–162.
Morrison, R.B., Pijoan, C., Hilley, H.D., Rapp, V., 1985. Microorganisms associated with
pneumonia in slaughter weight swine. Can. J. Comp Med. 49, 129-137.
58
CHAPTER 1: GENERAL INTRODUCTION
Mul, M., Vermeij, I., Hindle, V., Spoolder, H., 2010. EU-welfare legislation on pigs.
Wageningen UR Livestock Research. Report 273, 1-34.
Muneta, Y., Minagawa, Y., Shimoji, Y., Nagata, R., Markham, P., Browning, G., Mori, Y., 2006.
IL-18
expression
in
pigs
following
infection
with
Mycoplasma
hyopneumoniae.
J. Interferon Cytokine Res. 26, 637-644.
Muneta, Y., Minagawa, Y., Shimoji, Y., Ogawa, Y., Hikono, H., Mori, Y., 2008. Immune
response of gnotobiotic piglets against Mycoplasma hyopneumoniae. J. Vet. Med. Sci. 70, 10651070.
Murphy, D., Van Alstine, W.G., Clark, L.K., Albregts, S., Knox, K., 1993. Aerosol vaccination
of pigs against Mycoplasma hyopneumoniae infection. Am. J. Vet. Res. 54, 1874-1880.
Murtaugh, I., Foss, M., 2002, Inflammatory cytokines and antigen presenting cell activation, Vet.
Immunol. and Immunopathol. 87, 109–121.
Ogawa, Y., Oishi, E., Muneta, Y., Sano, A., Hikono, H., Shibahara, T., Yagi, Y., Shimoji, Y.,
2009. Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix
rhusiopathiae vaccine strain as a vector. Vaccine 27, 4543-4550.
Okada, M., Sakano, T., Senna, K., Maruyama, T., Murofushi, J., Okonogi, H., Sato, S., 1999.
Evaluation of Mycoplasma hyopneumoniae inactivated vaccine in pigs under field conditions. J.
Vet. Med. Sci. 61, 1131-1135.
Opriessnig, T., Thacker, E.L., Yu, S., Fenaux, M., Meng, X.J., Halbur, P.G., 2004. Experimental
reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with
Mycoplasma hyopneumoniae and porcine circovirus type 2. Vet. Pathol. 41, 624-640.
Ostanello, F., Dottori, M., Gusmara, C., Leotti, G., Sala, V., 2007. Pneumonia disease
assessment using a slaughterhouse lung-scoring method. J. Vet. Med. A Physiol Pathol. Clin.
Med. 54, 70-75.
59
CHAPTER 1: GENERAL INTRODUCTION
Otagiri, Y., Asai, T., Okada, M., Uto, T., Yazawa, S., Hirai, H., Shibata, I., Sato, S., 2005.
Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested
PCR and culture methods. J. Vet. Med. Sci. 67, 801-805.
Otake, S., Dee, S., Corzo, C., Oliveira, S., Deen, J., 2010. Long distance airborne transport of
infectious PRRSV and Mycoplasma hyopneumoniae from a swine population infected with
multiple viral variants. Vet. Microbiol.. 145, 198-208
Palzer, A., 2006. Keimspektrum und Erregerassoziationen bei gesunden und an Pneumonie
erkrankten Schweinen. Ludwig-Maximilians-University Munich, Munich, Germany, Thesis, 56.
Park, S.C., Yibchok-Anun, S., Cheng, H., Young, T.F., Thacker, E.L., Minion, F.C., Ross, R.F.,
Hsu, W.H., 2002. Mycoplasma hyopneumoniae increases intracellular calcium release in porcine
ciliated tracheal cells. Infect. Immun. 70, 2502-2506.
Pieters, M., Pijoan, C., Fano, E., Dee, S., 2009. An assessment of the duration of Mycoplasma
hyopneumoniae infection in an experimentally infected population of pigs. Vet. Microbiol. 134,
261-266.
Piffer, I, Ross, R.F., 1984. Effect of age on susceptibility of pigs to Mycoplasma hyopneumoniae
pneumonia. Am. J. Vet. Res. 45, 478–481.
Pollack, J. D., 2002. Central carbohydrate pathways: metabolic flexibility and the extra role of
some ‗‗housekeeping‘‘ enzymes. In Molecular Biology and Pathogenicity of Mycoplasmas,
Edited by S. Razin & R. Herrmann. New York: Kluwer. pp. 163–201.
Raccach, M., Rottem, S., Razin, S., 1975. Survival of frozen Mycoplasmas. Applied Microb. 30,
167-171
Rautiainen E, Tuovinen V, Levonen K., 2000a. Monitoring antibodies to Mycoplasma
hyopneumoniae in sow colostrum--a tool to document freedom of infection. Acta Vet. Scand.
41, 213-25.
60
CHAPTER 1: GENERAL INTRODUCTION
Rautiainen, E., Virtala, A.M., Wallgren, P., Saloniemi, H., 2000b. Varying effects of infections
with Mycoplasma hyopneumoniae on the weight gain recorded in three different multisource
fattening pig herds. J. Vet. Med. B Infect. Dis. Vet. Public Health 47, 461-469.
Rautiainen, E., Oravainen, J., Virolainen, J.V., Tuovinen, V., 2001a. Regional eradication of
Mycoplasma hyopneumoniae from pig herds and documentation of freedom of the disease. Acta
Vet. Scand. 42, 355-364.
Rautiainen, E., Wallgren, P., 2001b. Aspects of the transmission of protection against
Mycoplasma hyopneumoniae from sow to offspring. J. Vet. Med. B Infect. Dis. Vet. Public
Health 48, 55-65.
Razin, S., Yogev, D., Naot, Y., 1998. Molecular biology and pathogenicity of Mycoplasmas.
Microbiol MolBiol Rev. 62, 1094-1156.
Redondo, E., Masot, A.J., Fernandez, A., Gazquez, A., 2009. Histopathological and
immunohistochemical findings in the lungs of pigs infected experimentally with Mycoplasma
hyopneumoniae. J. Comp Pathol. 140, 260-270.
Rice, P., Houshaymi, B.M., Abu-Groun, E.A., Nicholas, R.A. and Miles, R.J., 2001. Rapid
screening of H2O2 production by Mycoplasma mycoides and differentiation of European subsp.
mycoides SC (small colony) isolates. Vet. Microbiol. 78:343-351.
Rodriguez,
F.,
Ramirez,
G.A.,
Sarradell,
J.,
Andrada,
M.,
Lorenzo,
H.,
2004.
Immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with
Mycoplasma hyopneumoniae. J. Comp. Pathol. 130, 306-312.
Rodriguez,
F.,
Quesada,
O.,
Poveda,
J.B.,
Fernandez,
A.,
Lorenzo,
H.,
2007.
Immunohistochemical detection of interleukin-12 and interferon-gamma in pigs experimentally
infected with Mycoplasma hyopneumoniae. J. Comp. Pathol. 136, 79-82.
Roman, A.V., Lukesova, D., Novak, P., Zizlavsky, M., 2006. Biosecurity in pig herds.
Agricultura tropica e subtropica 39, 119-124.
61
CHAPTER 1: GENERAL INTRODUCTION
Roof, M.B., Burkhart, K., Zuckermann, F., 2001. Evaluation of the immune response and
efficacy of 1 and 2 dose commercial Mycoplasma hyopneumoniae bacterins. In: Proceedings of
the 32nd Annual Meeting of the American Association of Swine Veterinarians, Nashville,
Tennessee, 163– 167.
Rose, N., Madec, F., 2002. Occurrence of respiratory disease outbreaks in fattening pigs:
Relation with the features of a densely and a sparsely populated pig area in France. Vet. Res. 33,
179–190
Rosendal, S., Mitchell, W. R., 1983. Epidemiology of Haemophilus pleuropneumoniae infection
in pigs. Can. J. Comp. Med. 47, 1–5.
Ross, R., 1999. Mycoplasmal diseases. In: Leman, A.D., Straw, B.E., D‘Allaire, S., Mengeling,
W.L., and Taylor, D.J., (Eds), Diseases of Swine, 8th edn. The Iowa State University Press,
Ames, IA, pp 495-509.
Ross, R.F., Zimmermann-Erickson, B.J., Young, T.F., 1984. Characteristics of protective activity
of Mycoplasma hyopneumoniae vaccine. Am. J. Vet. Res. 45, 1899-1905.
Ross, R. F., 1999. Mycoplasmal diseases, In B. E. Straw, S. D‘allaire, W. L. Mengeling, and D.
J. Taylor (ed.), Diseases of swine, 8th ed. Iowa State University Press, Ames, Iowa.p. 537–551.
Ruiz, A., Utrera, V., Pijoan, C., 2003. Effect of Mycoplasma hyopneumoniae sow vaccination on
piglet colonization at weaning. J. Swine Health Prod. 11, 131-135.
Sarradell, J., Andrada, M., Ramirez, A.S., Fernandez, A., Gomez-Villamandos, J.C., Jover, A.,
Lorenzo, H., Herraez, P., Rodriguez, F., 2003. A morphologic and immunohistochemical study
of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma
hyopneumoniae. Vet. Pathol. 40, 395-404.
62
CHAPTER 1: GENERAL INTRODUCTION
Sheldrake, R.F., Gardner, I.A., Saunders, M.M. and Romalis, L.F., 1990. Serum antibody
response to Mycoplasma hyopneumoniae measured by enzyme-linked immunosorbent assay
after experimental and natural infection of pigs. Aus. Vet. J. 67, 39–42.
Sheldrake, R.F., Gardner, I.A., Saunders, M.M., Romalis, L.F., 1991. Intraperitoneal vaccination
of pigs to control Mycoplasma hyopneumoniae. Res. Vet. Sci. 51, 285-291.
Sheldrake, R.F., Romalis, L.F., Saunders, M.M., 1993. Serum and mucosal antibody responses
against Mycoplasma hyopneumoniae following intraperitoneal vaccination and challenge of pigs
with M hyopneumoniae. Res. Vet. Sci. 55, 371-376.
Shimoji, Y., Oishi, E., Muneta, Y., Nosaka, H., Mori, Y., 2003. Vaccine efficacy of the
attenuated Erysipelothrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma
hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine. Vaccine 21, 532-537.
Sibila, M., Calsamiglia, M., Vidal, D., Badiella, L., Aldaz, A., Jensen, J.C., 2004a. Dynamics of
Mycoplasma hyopneumoniae infection in 12 farms with different production systems. Can. J.
Vet. Res. 68, 12-18.
Sibila, M., Calsamiglia, M., Segales, J., Rosell, C., 2004b. Association between Mycoplasma
hyopneumoniae at different respiratory sites and presence of histopathological lung lesions. Vet.
Rec. 155, 57-58.
Sibila, M., Nofrarias, M., Lopez-Soria, S., Segales, J., Riera, P., Llopart, D., Calsamiglia, M.,
2007a. Exploratory field study on Mycoplasma hyopneumoniae infection in suckling pigs. Vet.
Microbiol. 121, 352-356.
Sibila, M., Nofrarias, M., Lopez-Soria, S., Segales, J., Valero, O., Espinal, A., Calsamiglia, M.,
2007b. Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and
associated lung lesions in vaccinated and non-vaccinated pigs. Vet. Microbiol. 122, 97-107.
Sibila, M., Bernal, R., Torrents, D., Riera, P., Llopart, D., Calsamiglia, M., Segales, J., 2008.
Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization
and seroconversion, and pig lung lesions at slaughter. Vet. Microbiol. 127, 165-170.
63
CHAPTER 1: GENERAL INTRODUCTION
Sibila, M., Pieters, M., Molitor, T., Maes, D., Haesebrouck, F., Segales, J., 2009. Current
perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection. Vet.
J. 181, 221-231.
Silva, L.A., Castro, A., Silva Júnior, M.P., Moraes, M.A.S. , Moreira, M.R., 2009. Detection of
Mycoplasma hyopneumoniae in lungs and nasal swabs ofpigs by nested PCR. Almeida Arq.
Bras. Med. Vet. Zootec. 61, 149-155.
Simionatto, S., Marchioro, SB., Galli, V., Hartwig, D.D., Carlessi, R.M., Munari, F.M., Laurino,
J.P., Conceição, F.R., Dellagostin, O.A., 2010. Cloning and purification of recombinant proteins
of Mycoplasma hyopneumoniaeexpressed in Escherichia coli. Protein Expr Purif 2, 132-136.
Sørensen, V., Ahrens, P., Barfod, K., Feenstra, A.A., Feld, N.C., Friis, N.F., Bille-Hansen, V.,
Jensen, N.E., Pedersen, M.W., 1997. Mycoplasma hyopneumoniae infection in pigs: duration of
the disease and evaluation of four diagnostic assays. Vet. Microbiol. 54, 23-34.
Stakenborg, T., Vicca, J., Maes, D., Peeters, J., de, K.A., Haesebrouck, F., Butaye, P., 2006.
Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates. J.
Microbiol. Methods 66, 263-275.
Stanković, B., Hristov, S., Bojkovski, T. J., Maksimović, N., 2010. Health status and bio-security
plans on pig farms. Biotechnology in Animal Husbandry 26, 29-35.
Stärk, K., Keller, H. and Eggenberger, E., 1992. Risk factors for the reinfectionof specific
pathogen-free pig breeding herds with enzootic pneumonia. Vet. Rec., 131, 532-535.
Stärk, K.D., Nicolet, J., Frey, J., 1998a. Detection of Mycoplasma hyopneumoniae by air
sampling with a nested PCR assay. Appl Environ Microbiol. 25, 327-330.
Stärk, K., 1998b. Systems for the prevention and control of infectious diseases in pigs. Massey
University, Palmerston North, New Zealand. Thesis p. 9-27.
Stärk, K., 2000. Epidemiological investigation of the influence of environmental risk factors on
respiratory diseases in swine — A literature review. Vet. J. 159 , 37–56.
64
CHAPTER 1: GENERAL INTRODUCTION
Stipkovits, L., Miller, D., Glavits, R., Fodor, L., Burch, D., 2001. Treatment of pigs
experimentally infected with Mycoplasma hyopneumoniae, Pasteurella multocida, and
Actinobacillus pleuropneumoniae with various antibiotics. Can. J. Vet. Res. 65, 213-222.
Strait, E.l., Erickson B.Z., Thacker, E.l., 2004. Analysis of Mycoplasma hyopneumoniae field
isolates. In Proceedings of AASV Congress, Des Moines, Iowa. 95-96.
Strait, E.L., Madsen, M.L., Minion, F.C., Christopher-Hennings, J., Dammen, M., Jones, K.R.,
Thacker, E.L., 2008. Real-time PCR assays to address genetic diversity among strains of
Mycoplasma hyopneumoniae. J. Clin. Microbiol. 46, 2491-2498.
Strasser, M., Abiven, P., Kobisch, M., Nicolet, J., 1992. Immunological and pathological
reactions in piglets experimentally infected with Mycoplasma hyopneumoniae and/or
Mycoplasma flocculare. Vet. Immunol. Immunopathol. 31, 141-153.
Strasser, M., Frey, J., Bestetti, G., Kobisch, M., Nicolet, J., 1991. Cloning and expression of a
species-specific early immunogenic 36-kilodalton protein of Mycoplasma hyopneumoniae in
Escherichia coli. Infect. Immun. 59, 1217-1222.
Suter, M., Kobisch, M., Nicolet, J., 1985. Stimulation of immunoglobulin-containing cells and
isotype-specific antibody response in experimental Mycoplasma hyopneumoniae infection in
specific-pathogen-free pigs. Infect. Immun. 49, 615-620.
Tajima, M., Yagihashi, T., 1982. Interaction of Mycoplasma hyopneumoniae with the porcine
respiratory epithelium as observed by electron microscopy. Infect. Immun. 37, 1162-1169.
Talaat , A.M. & Stemke-Hale, K., 2005. Expression library immunization: a road map for
discovery of vaccines against infectious diseases. Infect Immun 73, 7089–7098.
Taylor, R.B., Titer,P, Manzo, C., 1979. Immunoregulatory effects of a covalent antgen-antibody
complex. Nature, 281, 488.
65
CHAPTER 1: GENERAL INTRODUCTION
Thacker, E.L., Thacker, B.J., Kuhn, M., Hawkins, P.A., Waters, W.R., 2000. Evaluation of local
and systemic immune responses induced by intramuscular injection of a Mycoplasma
hyopneumoniae bacterin to pigs. Am. J. Vet. Res. 61, 1384-1389.
Thacker, E.L., Thacker, B.J., Janke, B.H., 2001. Interaction between Mycoplasma
hyopneumoniae and swine influenza virus. J. Clin. Microbiol. 39, 2525-2530.
Thacker, E.L., Thanawongnuwech , R., 2002. Porcine Respiratory Disease Complex (PRDC).
Thai J. Vet. Med. 32, 126-134.
Thacker, E.L., 2004. Diagnosis of Mycoplasma hyopneumoniae. Anim Health Res. Rev. 5, 317320.
Thacker, E.L., 2006. Mycoplasmal diseases. In: Leman, A.D., Straw, B.E., D‘Allaire, S.,
Mengeling, W.L., and Taylor, D.J., (Ed.), Diseases of Swine, 9th ed. The Iowa State University
Press, Ames, IA, pp 701-717.
Thanawongnuwech, R., Young, T.F., Thacker, B.J., Thacker, E.L., 2001. Differential production
of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection
model. Vet. Immunol. Immunopathol. 79 , 115-127.
Thanawongnuwech, R., Thacker, E.L., 2003. Interleukin-10, interleukin-12, and interferongamma levels in the respiratory tract following Mycoplasma hyopneumoniae and PRRSV
infection in pigs. Viral Immunol. 16, 357-367.
Thanawongnuwech, R., Thacker, B., Halbur, P., Thacker, E.L., 2004. Increased production of
proinflammatory cytokines following infection with porcine reproductive and respiratory
syndrome virus and Mycoplasma hyopneumoniae. Clin. Diagn. Lab Immunol. 11, 901-908.
Thomsen, B.L., Jorsal, S.E., Andersen, S., Willeberg, P., 1992. The Cox regression model
applied to risk factor analysis of infections in the breeding and multiplying herds in the Danish
SPF system. Prev Vet Med. 12, 287-297.
66
CHAPTER 1: GENERAL INTRODUCTION
Timmerman, T., Dewulf, J. , Catry, B., Feyen, Opsomer, B. G. , de Kruif A., and Maes, D.,
2006. Quantification and evaluation of antimicrobial-drug use in group treatments for fattening
pigs in Belgium, Prev. Vet. Med. 74, 251–263.
Turek, J.J., Schoenlein, I.A., Watkins, B.A., Van Alstine, W.G., Clark, L.K., Knox, K., 1996.
Dietary polyunsaturated fatty acids modulate responses of pigs to Mycoplasma hyopneumoniae
infection. J. Nutr. 1541-1547
Vasconcelos, A.T., Ferreira, H.B., Bizarro, C.V. et., 2005. Swine and poultry pathogens: the
complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of
Mycoplasma synoviae. J. Bacteriol. 187, 5568-5577.
Verdin, E., Kobisch, M., Bove, J.M., Garnier, M., Saillard, C., 2000a. Use of an internal control
in a nested-PCR assay for Mycoplasma hyopneumoniae detection and quantification in
tracheobronchiolar washings from pigs. Mol. Cell Probes 14, 365-372.
Verdin, E., Saillard, C., Labbe, A., Bove, J.M., Kobisch, M., 2000b. A nested PCR assay for the
detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs. Vet.
Microbiol. 76, 31-40.
Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F., de, K.A., 2002. Patterns of
Mycoplasma hyopneumoniae infections in Belgian farrow-to-finish pig herds with diverging
disease-course. J. Vet. Med. B Infect. Dis. Vet. Public Health 49, 349-353.
Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de, K.A., Haesebrouck, F., 2003.
Evaluation of virulence of Mycoplasma hyopneumoniae field isolates. Vet. Microbiol. 97, 177190.
Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de, K.A., Haesebrouck, F., 2004. In
vitro susceptibilities of Mycoplasma hyopneumoniae field isolates. Antimicrob. Agents
Chemother. 48, 4470-4472.
67
CHAPTER 1: GENERAL INTRODUCTION
Vicca, J., 2005a. Virulence and antimicrobial susceptibility of Mycoplasma hyopneumoniae
isolates from pigs. Faculty of Veterinary Medicine. Gent University. Thesis 11-37.
Vicca, J., Maes, D., Jonker, L., de, K.A., Haesebrouck, F., 2005b. Efficacy of in-feed medication
with tylosin for the treatment and control of Mycoplasma hyopneumoniae infections. Vet. Rec.
156, 606-610.
Vicca, J., Maes, D., Stakenborg, T., Butaye, P., Minion, F., Peeters, J., de, K.A., Decostere, A.,
Haesebrouck, F., 2007. Resistance mechanism against fluoroquinolones in Mycoplasma
hyopneumoniae field isolates. Microb. Drug Resist. 13, 166-170.
Walker, J., Lee, R., Mathy, N., Doughty, S., Conlon, J., 1996. Restricted B-cell responses to
microbial challenge of the respiratory tract. Vet. Immunol. Immunopathol. 54, 197-204.
Wallgren, P., Artursson, K., Fossum, C., Alm, G.V., 1993a. Incidence of infections in pigs bred
for slaughter revealed by elevated serum levels of interferon and development of antibodies to
Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae. Zentralbl. Veterinarmed. B
40, 1-12.
Wallgren, P., Sahlander, P., Hassleback, G., Heldmer, E., 1993b. Control of infections with
Mycoplasma hyopneumoniae in swine herds by disrupting the chain of infection, disinfection of
buildings and strategic medical treatment. Zentralbl. Veterinarmed. B 40, 157-169.
Walter, D., Holck, J.T., Sornsen, S., Hagen, C., Harris, I.T., 2000. The effect of a metaphylactic
pulse dosing in feed antimicrobial strategy on finishing pig health and performance. Swine
Health Prod. 8, 65-71.
Whittlestone, P., 1973. Enzootic pneumonia of pigs (EPP). Adv. Vet. Sci. Comp. Med. 17, 1-55.
Whittlestone, P., 1976. Immunity to mycoplasmas causing respiratory diseases in man and
animals. Adv. Vet. Sci. Comp. Med., 20, 277-307.
68
CHAPTER 1: GENERAL INTRODUCTION
Winner, F., Rosengarten, R. and Citti, C., 2000. In vitro cell invasion of Mycoplasma
gallisepticum. Infect. Immun. 68, 4238-4244.
Woeste, K., grosse Beilage, E., 2007. Transmission of agents of the porcine respiratory disease
complex (PRDC) between swine herds: a review. Part 2--Pathogen transmission via semen, air
and living/nonliving vectors. Dtsch. Tierarztl. Wochenschr. 114, 364-373.
Wu, C., Shryock, T., Lin, T., Venhuizen, M., 1997. Testing antimicrobial susceptibility against
Mycoplasma hyopneumoniae in vitro. 1997. Swine Health Prod. 5, 227-230.
Yeske, P., 2001. Experiences with mycoplasma vaccinations: what to do if vaccination doesn't
live up to expectations. Proceedings of the Allen D. Leman Conference University of Minnesota,
USA, pp 108-110.
Zhang, Q., Young, T.F., Ross, R.F., 1995. Identification and characterization of a Mycoplasma
hyopneumoniae adhesin. Infect. Immun. 63, 1013-1019.
Zhuang, Q., Wachmann, H., Mortensen, S., Barford, K., 2002. Incidence of Actinobacillus
pleuropneumoniae serotype 2 and Mycoplasma hyopneumoniae infections in the Danish SPF
herds and risk factors for infections. In: Proceedings of the 17th International Pig Veterinary
Society, Ames, Iowa. p. 228.
Zielinski, G.C., Ross, R.F., 1993. Adherence of Mycoplasma hyopneumoniae to porcine ciliated
respiratory tract cells. Am. J. Vet. Res. 54, 1262-1269.
Zimmermann, W., Odermatt, W., Tschudi, P., 1989. Enzootic Pneumonia (EP): The partial
depopulation of sow farms reinfected with EP as an alternative to the complete depopulation.
Schweiz Arch Tierheilk, 179-191.
69
CHAPTER 2: AIMS OF THE STUDY
70
CHAPTER 2: AIMS OF THE STUDY
CHAPTER 2.
AIMS OF THE STUDY
71
CHAPTER 2: AIMS OF THE STUDY
AIMS OF THE STUDY
M. hyopneumoniae is the primary agent of enzootic pneumonia, a chronic respiratory disease in
pigs that occurs worldwide and causes major economic losses. The clinical course of infections
may vary significantly between pig herds depending on herd management strategies and
environmental conditions. However, also the virulence of the M. hyopneumoniae strains may
determine the clinical outcome, which renders control of this pathogen a difficult task. Control
measures include the optimization of management and housing conditions, the use of strategic
medication and/or vaccination. The current control measures are beneficial from an economic
point of view, but they do not entail a sustainable solution for the disease.
There is limited information on the occurrence and risk factors involved in early infection and
there are still many questions regarding the effect of vaccination on transmission of this
pathogen. The limited studies available have mainly focused on experimental conditions and do
not necessarily reflect the situation in the field. In addition, information on differences between
infection course caused by low and highly virulent field strains and the effect of vaccination on
infection with different M. hyopneumoniae strains is very scarce. Answers to these questions are
necessary for a better understanding of the pathogenicity of this microorganism, and to improve
the control of enzootic pneumonia.
The general aim of this study was to assess the importance of early infection and transmission of
M. hyopneumoniae under field conditions and to investigate protection against different M.
hyopneumoniae strains.
The specific objectives were:
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CHAPTER 2: AIMS OF THE STUDY
1. To estimate the detection rate of M. hyopneumoniae infections in piglets at 3 weeks of
age in different European countries by analysing nasal swabs using nPCR,
2. To quantify the transmission of M. hyopneumoniae in nursery pigs under field conditions,
and to assess the effect of vaccination,
3. To assess the efficacy of a commercial vaccine against infection with M. hyopneumoniae
strains of low and high virulence,
4. To investigate the infection pattern and lung lesion development in pigs experimentally
infected with low and highly virulent M. hyopneumoniae strains,
5. To investigate the effect of infection with low virulent M. hyopneumoniae strains on a
subsequent challenge infection with a highly virulent strain.
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CHAPTER 2: AIMS OF THE STUDY
74
CHAPTER 3: EXPERIMENTAL STUDIES
CHAPTER 3.
EXPERIMENTAL STUDIES
75
CHAPTER 3: EXPERIMENTAL STUDIES
76
CHAPTER 3: EXPERIMENTAL STUDIES
Early infection and transmission of Mycoplasma
hyopneumoniae under field conditions
77
CHAPTER 3: EXPERIMENTAL STUDIES
78
CHAPTER 3: EXPERIMENTAL STUDIES
3.1 Early Mycoplasma hyopneumoniae infections in suckling pigs in herds with
respiratory problems in Europe: detection rate and risk factors
Iris Villarreal a,*, Katleen Vranckx a, Luc Duchateau a, Frank Pasmans a, Freddy Haesebrouck a,
Jens C. Jensen b, Ian A. Nanjiani c, Dominiek Maes a
a
Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke,
Belgium
b
c
Orion Pharma Animal Health, Møllevej 9A, 2990 Nivå, Denmark
Pfizer Ltd. VMRD, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
Veterinarni Medicina, 55, 2010 (7): 318–324
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CHAPTER 3: EXPERIMENTAL STUDIES
Abstract
The present study aimed to estimate the detection rate of M. hyopneumoniae in 3-week-old pigs
in different European countries and to identify possible risk factors. Nasal swabs from suckling
pigs in 52 farms were collected for analysis using nested PCR. Potential risk factors for
respiratory disease were analysed with a multivariable logistic regression model. The average
percentage of positive piglets was 10.7% (95% confidence interval, CI 7.4-14.2); at least one pig
tested positive in 68% of herds. In 32% of the herds, more than 10% of piglets tested positive.
Herds that vaccinated sows against swine influenza virus (SIV) had a significantly higher risk of
a piglet being positive for M. hyopneumoniae (OR 3.12; 95% CI 1.43-6.83). The higher risk in
case of SIV vaccination is difficult to explain, but it may be due to the fact that pig herds with
respiratory symptoms are more likely to be vaccinated against SIV, overlooking the possible
influence of other respiratory pathogens such as M. hyopneumoniae. The present findings show
that M. hyopneumoniae is widespread in 3-week-old piglets across different European countries.
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CHAPTER 3: EXPERIMENTAL STUDIES
1. Introduction
Infections with Mycoplasma hyopneumoniae occur worldwide and cause major economic losses
to the pig industry. Control of M. hyopneumoniae can be accomplished by optimising
management and housing practices, by antimicrobial medication and by vaccination. In many
European countries, more than half of the pig herds practice vaccination to control the infection
(Maes et al., 2008). Different vaccination protocols are used, but in most instances the piglets are
vaccinated once or twice in the farrowing unit or at weaning.
The spread of M. hyopneumoniae occurs by either vertical transmission from the sow to the
offspring (Sibila et al., 2007) or by horizontal transmission between pigs (Meyns et al., 2004;
Fano et al., 2007). Sows transmit M. hyopneumoniae to the offspring mainly by direct nose-tonose contact, as there is no transmission via the intra-uterine route, nor by colostrum or milk.
The importance of the sow for infection of the piglets is not clear and has been investigated only
in a few studies including a limited number of pig herds. Goodwin et al. (1965) reported that
younger sows are more likely to transmit M. hyopneumoniae to their piglets than older sows.
Older sows, especially these in endemically infected herds, have a lower probability of
harbouring M. hyopneumoniae in their respiratory tract (Clark et al., 1991). Calsamiglia and
Pijoan (2000) found a higher percentage of young sows infected with M. hyopneumoniae in their
upper respiratory tract, but the microorganism was also demonstrated in nasal samples of older
sows (3rd to 7th parity).
Studies using nested PCR (nPCR) on nasal swabs from piglets before weaning in a limited
number of herds have been performed. Calsamiglia and Pijoan (2000) tested animals in a three81
CHAPTER 3: EXPERIMENTAL STUDIES
site system and found 7.7-9.6% positive pigs at 17 days of age. Ruiz et al. (2003) found 5.513.2% of piglets positive on a breeding farm at 19 days of age. Sibila et al. (2007) performed
nasal sampling in piglets from 1 and 3 weeks of age; the percentage of positive piglets ranged
between 0 and 6.4%. Much higher infection rates were obtained by Fano et al. (2007). They
reported a prevalence of 2.5-51.8% in piglets at one day before weaning (17 days of age), in 11
batches of a multi-site production farm. Although the presence of DNA in nasal swabs is not a
100% guarantee for infection, it is certainly an indication that these animals were exposed to the
organism.
Sibila et al. (2007) performed nPCR on bronchial and tonsillar swabs of 37 pigs at 3 weeks of
age and they showed the presence of mycoplasmal DNA in the tonsils of one pig and in the
bronchial swab of two other pigs. Infection of piglets during the suckling period is particularly
important, since piglets are transferred to other pens and are usually regrouped with other piglets
at weaning. In this way, infected piglets may easily transmit the infection to other non-infected
pigs during nursery.
To implement appropriate control measures and to further optimise the vaccination strategies, it
is important to have a precise notion of how widespread this agent is in young pigs from
different pig herds in different countries. Knowledge on early infection is particularly important
for the European pig industry, as early vaccination is widely practised (Maes et al., 2003) and a
major part of the industry are farrow-to-finish pig herds. Apart from assessing the infection rate,
it is also important to identify possible herd factors that may predispose to early infections in
pigs. The aim of the present study was to estimate the detection rate of M. hyopneumoniae
infections in piglets at 3 weeks of age in different European countries by analysing nasal swabs
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using nPCR. In addition, possible risk factors associated with M. hyopneumoniae infection in
young piglets were investigated.
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CHAPTER 3: EXPERIMENTAL STUDIES
2. Materials and methods
Study herds
The study was conducted from May 2008 to March 2009 in nine European countries (Belgium,
Denmark, France, Germany, Hungary, Italy, Poland, Spain and The Netherlands). The target
population consisted of 52 pig herds. In each country, 6 single-site farrow-to-finish or sow herds
were included, except for Germany, where 4 herds were included. The herds had to comply with
specific selection criteria, such as a minimum herd size of 100 sows, presence of clinical
respiratory problems related to M. hyopneumoniae (coughing in grower-finishing pigs) and no
use of antimicrobials active against M. hyopneumoniae in piglets less than 3 weeks of age.
Nasal swabs
To obtain with 95% certainty a detection rate of at least 10% in the selected countries, a
minimum of 30 piglets had to be sampled per herd. Hence, 30 nasal swabs were collected in each
herd from piglets of 21 ± 3 days of age. The piglets within each herd were selected randomly
from as many different sows as possible. In case there were less than 30 mother sows, two
piglets per sow were selected.
Nasal swabs were obtained by deeply swabbing the nasal mucosa of one nostril using a cottontipped swab. The samples from each country were stored at -70 °C.
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CHAPTER 3: EXPERIMENTAL STUDIES
Analysis of nasal samples
The material collected in each nasal swab was suspended in 200 µL phosphate buffered saline
(PBS) and DNA was extracted using the Qiagen, DNAeasy Blood & Tissue Kit). To detect M.
hyopneumoniae DNA, a nPCR protocol was used (Villarreal et al., 2009). The nested PCR
reactions were performed using Taq polymerase. In the first step, a final volume of 20 μL
contained 2 μL 10x PCR buffer, 2 μL 3 mM MgCl2, 0.36 μL 170 μM dNTPs, 12.5 μL of high
performance liquid chromatography water (HPLC-H2O), 0.1 μL 0.03 U/μL Taq polymerase
(Gibco Invitrogen), 0.5 μL each primer MHP950-1L (5‘-AggAACACCATCgCgATTTTTA-3‘)
and MHP950-1R (5‘-ATAAAAATggCATTCCTTTTCA-3‘) and 2 μL 10-fold-diluted DNA
solution.
In the second step, a final volume of 15 μL contained 1.5 μL 10x PCR buffer, 0.9 μL 3 mM
MgCl2, 0.27 μL 170 μM dNTPs, 10 μL HPLC-H2O, 0.08 μL 0.03 U/μL Taq polymerase, 0.5 μL
primers
MHP950-2L
(5‘-CCCTTTgTCTTAATTTTTgCAA-3‘)
and
MHP950-2R
(5‘-
gCCgATTCTAgTACCCTAATCC-3‘) and 1.5 μL 10-fold-diluted DNA solution.
The following PCR programme was used for both steps: initial denaturation at 94 °C for 2 min;
denaturation at 94 °C for 30 s, annealing at 51 °C for 30 s and extension at 72 °C for 1 min; 35
cycles of denaturation at 94 °C for 20 s, annealing at 55 °C for 1 min and extension at 72 °C for
1 min; final extension at 72 °C for 5 min, followed by cooling to 10 °C. Nested PCR products
were analysed by electrophoresis on 1.5% agarose gels in Tris-Borate-EDTA (TBE) buffer and
visualised under ultraviolet illumination after staining with GelRed (Biotium). Positive and
negative controls were used for both DNA extraction and amplification.
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CHAPTER 3: EXPERIMENTAL STUDIES
Herd data collection
A questionnaire related to respiratory disease and herd management practices was completed
during a face-to-face interview with the farmer and when inspecting the sheds for each sampled
herd. Thirty eight questions were included in the analysis. To ensure standardisation of the
answers, most of these questions were closed (28) or semi-closed (10). The questionnaire was
pretested in three pig herds before the start of the study. The questions were divided in six
categories such as general herd information, sow population, purchase policy, housing
conditions, feeding management and health control strategies. For logistical and language
reasons, the completion of the questionnaires and the sampling were performed by the herd
veterinarians in each country.
Statistical analyses
The detection rate in each herd was based on the number of positive nasal swabs detected by
nPCR. In order to determine the risk factors associated with the detection rate of M.
hyopneumoniae at herd level, the PROC GLIMMIX procedure (SAS 9.2, 2008) was used to fit a
generalised mixed model for the presence of M. hyopneumoniae with country and herd nested in
country as random effects and binomially distributed error term. The predictor variables with P
≤0.10 in the univariate models were subsequently included in the multivariable analysis. A
backward likelihood ratio selection criterion was used for the multivariable regression model in
order to choose the variables that were significantly associated with the outcome variable (P
≤0.05) at the multivariable level. At each step, a variable with the highest P value (and >0.05)
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CHAPTER 3: EXPERIMENTAL STUDIES
was excluded from the model until no additional variables could be removed. One-way
biologically plausible interactions between the independent variables were tested in the final
model. The goodness-of-fit was evaluated by the ratio of the χ2 statistic and the corresponding
degrees of freedom with a value close to 1. Results were summarised using odd ratios with their
95% confidence intervals.
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CHAPTER 3: EXPERIMENTAL STUDIES
3. Results
In total, 52 herds and 1,555 piglets were sampled for presence of M. hyopneumoniae. Of all
herds included in the study, 28.6% were farrow-to-finish herds, 26.5% and 24.5% were sow
herds from which the piglets were moved to another farm after weaning or at 20-25 kg,
respectively, and the remainder of the herds (20.4%) raised a part of the fattening pigs on the
same site and partly moved pigs after weaning or at 10 weeks of age. The sows from the sampled
pigs had following parity distribution: 1st to 2nd parity: 39%; 3rd to 4th parity: 30%; 5th to 7th
parity: 27%; and >7th parity: 4%. Twenty eight percent of the herds vaccinated sows against M.
hyopneumoniae before their first gestation.
The total average percentage of positive pigs within the herds was 10.7% (95% CI 7.4-14.2). In
herds vaccinating against M. hyopneumoniae 8.4% (95% CI 7.6-9.1) were infected, whereas only
2.6% (95% CI 1.8-3.4) were infected in the herds not vaccinating against M. hyopneumoniae.
Twenty eight percent of the herds practised sow vaccination against M. hyopneumoniae before
their first gestation, accounting for 3.1% of the M. hyopneumoniae positive piglets. No
significant difference was found between these and the herds not practicing sow vaccination
(P>0.05).
The minimum within-herd detection rate was 0% and the maximum within-herd detection rate
was 37% (Fig. 1). The percentage of herds with at least one positive tested pig was 68% (95% CI
51.7-83.4).
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CHAPTER 3: EXPERIMENTAL STUDIES
Fig 1. Distribution of herds according to within-herd detection rate of M. hyopneumoniae in 3week old piglets. Fifty-two pig herds from different European countries were included; 30 piglets
per herd were sampled.
35
30
% Herds
25
20
15
10
5
0
0
1-10
11-20
21-30
31-40
Within-herd detection rate (%)
The number of positive herds and positive animals in the different countries are presented in
Table 1. France, The Netherlands, Italy and Germany were the countries with the highest number
of herds positive for M. hyopneumoniae. In France, 100% of the herds were positive. The highest
within-herd detection rates were found in Italy, France and Germany.
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CHAPTER 3: EXPERIMENTAL STUDIES
Table 1: Detection rate of M. hyopneumoniae in nasal swabs of 3-week old pigs in different EU
countries.
Number of positive
herds/Number of
Number (%) of positive
Minimum-maximum % of
Country
herds sampled
pigletsa
positive pigs
Belgium
4/6
6
(3.3)
0.0-10.0
Denmark
3/6
3
(1.7)
0.0-3.30
France
6/6
38
(21.1)
10.0-30.0
Germanyb
3/4
23
(18.7)
0.0-26.7
Hungary
3/6
18
(10.0)
0.0-30.0
Italy
5/6
40
(22.1)
0.0-36.7
Poland
4/6
17
(9.7)
0.0-26.7
Spain
2/6
9
(5.0)
0.0-20.0
The Netherlands
5/6
14
(7.7)
0.0-16.1
Total (%)
a
35/52 (67.3)
168/1555 (10.7)
0.0-36.7
Fifty-two herds were sampled; 30 piglets were sampled in each herd: 6 herds per country, 30
pigs per herd = 180 pigs per country (only 4 herds or 120 pigs in Germany). Only 25 samples
were taken from one herd in Poland.
b
Only 4 herds were sampled.
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CHAPTER 3: EXPERIMENTAL STUDIES
Three of 38 variables were significant in the univariate analysis (P ≤0.10), namely sow
vaccination against swine influenza (SIV), sow vaccination against atrophic rhinitis and the
presence of a slaughterhouse within a distance of 5 km from the herd (Table 2). In total, 27% and
28% of herds vaccinated their sows against SIV and atrophic rhinitis, respectively. In 10% of the
herds, a slaughterhouse was located within 5 km of the herd.
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CHAPTER 3: EXPERIMENTAL STUDIES
Table 2: Potential risk factors (P <0.10) associated at herd level with the detection rate of M.
hyopneumoniae in piglets of 3 weeks of age after univariate analysis.
Variables
Number Level
Number (%) of
Mean % of
of herds
herds per level
positive herds per
P value
level
Sow vaccination against
52
swine influenza
Sow vaccination against
52
atrophic rhinitis
Presence of slaughterhouse
within 5 km from pig herd
49
No
38 (73.1)
5.0
Yes
14 (26.9)
15.5
No
37 (71.2)
5.4
Yes
15 (28.2)
13.4
No
44 (89.8)
6.6
Yes
5 (10.2)
21.6
0.014
0.029
0.034
These three variables were subsequently used in the multivariable model. As presented in table 3,
only sow vaccination against SIV was significantly (P <0.05) and positively associated with the
prevalence of M. hyopneumoniae in the piglets (OR 3.12; 95% CI 1.43-6.83) when adjusted for
the other covariates. Vaccination against atrophic rhinitis was not significantly associated with
the prevalence of M. hyopneumoniae in piglets, but the factor was retained in the final model
because the P value was borderline not significant (P = 0.052). The goodness of fit statistic was
0.95, close to and not differing significantly from 1, demonstrating that this model describes the
data well.
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CHAPTER 3: EXPERIMENTAL STUDIES
Table 3: Risk factors (P <0.05) for being M. hyopneumoniae positive in 3-week-old piglets in
nine European countries in the final multivariable logistic regression modela
Variable
Sow vaccination against swine
influenza
Sow vaccination against
atrophic rhinitisb
Level
-coefficient
No
Yes
1.14
No
Yes
Odds ratio (95% CI)
P value
1
0.023
3.12 (1.43-6.83)
1
0.91
0.052
2.48 (0.86-5.45)
a
Generalized χ2; Degrees of freedom 0.95.
b
Variable was included in the final model, since the P value was borderline not significant.
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CHAPTER 3: EXPERIMENTAL STUDIES
4. Discussion
The present study assessed the detection rate of M. hyopneumoniae in piglets of 3 weeks of age
in different types of pig herds located in different European countries. The results showed that
there was evidence of M. hyopneumoniae infection in 3-week old piglets in two thirds of the
study herds. In most of the positive herds, the detection rate was between 1 and 10%. In some
herds, more than 30% of the piglets were positive. This suggests that, in the selected herds, early
infections with M. hyopneumoniae are important (Sibila et al., 2004, 2007; Fano et al., 2005). As
specific criteria were used to select the herds (clinical problems in grower-finishing pigs
attributed to this pathogen), the results may not be generalised to all pig herds within the EU, but
only to herds meeting the selection criteria. However, in terms of management and housing
conditions, the selected herds can be considered to be representative for many other pig herds in
the EU (Sibila et al., 2004; Beilage et al., 2009).
Nested PCR on nasal swabs was used to analyse the samples. This diagnostic tool is suitable for
detection of M. hyopneumoniae under field conditions in suckling pigs and nursery pigs (Otagiri
et al., 2005; Sibila et al., 2007), detecting 5.1 x 106 ng M. hyopneumoniae DNA or as few as 4
organisms/μL reaction mixture (Gebruers et al., 2008). To avoid false positive results due to
contamination in the nPCR, appropriate positive and negative controls were used during DNA
extraction and amplification. The results should be interpreted at the herd level, not at the
individual level (Otagiri et al., 2005).
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CHAPTER 3: EXPERIMENTAL STUDIES
M. hyopneumoniae attaches to and multiplies on the ciliated epithelium of the trachea, bronchi
and bronchioles (Zielinski et al., 1993). Consequently, the nose is not the primary site of
multiplication of M. hyopneumoniae organisms. It is therefore likely that the present results
underestimate the number of infected animals in the selected herds and that a higher detection in
recently weaned piglets would have been obtained from bronchoalveolar lavage fluid (BALF)
(Moorkamp et al., 2009). Taking BAL fluid is possible under field conditions, but it is less
practical and more time consuming than taking nasal swabs.
Serology would not work in the present study as it is not possible to discriminate between serum
antibodies following infection and maternally derived antibodies. In addition, it takes at least 4-8
weeks before contact infected pigs may show detectable serum antibodies (Feld et al., 1992).
Isolation of the bacterium is the gold standard for diagnosis of M. hyopneumoniae, but for the
present study this method is inappropriate at herd level. Lung tissue or BAL fluid should be used
instead of nasal swabs, nonetheless, these are very expensive, time consuming and special
medium is required.
Although sow vaccination against M. hyopneumoniae is not commonly practiced, twenty eight
percent of the sows in our study had been vaccinated before their first gestation. The detection
rate of M. hyopneumoniae in the piglets was not significantly different between herds practicing
sow vaccination and those not practicing vaccination. One could expect that vaccination in sows
decreases the number of M. hyopneumoniae organisms shed by the sow and increases the level of
maternal antibodies provided to the offspring. These factors could lead to a slight decrease in the
95
CHAPTER 3: EXPERIMENTAL STUDIES
number of nPCR positive pigs at weaning (Sibila et al., 2007). The absence of a difference in the
present study can be due to differences in vaccination strategies employed by the herds (e.g.
timing of vaccination, age of the sows at vaccination, type of vaccine) and the large variability in
serological response (30-100%) that may occur following sow vaccination (Thacker et al., 1998).
Herds vaccinating sows against SIV had a three times higher risk for having test-positive pigs
compared to herds not vaccinating against SIV. From a biological point of view, it is not only
unlikely, but also hard to explain that SIV vaccination would predispose to early M.
hyopneumoniae infections. The significant and positive association may be due to the fact that
herds with respiratory problems are more likely to vaccinate their sows against SIV. As most of
the respiratory problems are due to multiple infections and also to non-infectious factors
(Thacker et al., 2002), it is possible that in these herds, M. hyopneumoniae and/or other
pathogens are involved in respiratory problems in addition to SIV.
These results indicate that, before a vaccination program is implemented, it is imperative to have
a full picture of the problem in a herd and not to rely solely on the evaluation of the clinical
symptoms. Performance of additional laboratory examinations (e.g. postmortem examination of
dead animals, serology of different age groups) is required to investigate the different pathogens
involved in the problem before establishing an aetiological diagnosis. Otherwise, vaccination
programs may not be sufficient to control the main pathogen(s) in the herd.
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CHAPTER 3: EXPERIMENTAL STUDIES
Finally, some comments should be made with regard to the use of a cross-sectional study design
and the causality of the risk factor. Only one group of piglets from each herd was sampled.
Cross-sectional studies measure events at a particular time and therefore cause and effect are
sometimes difficult to separate. The risk factor found in this study did not fulfil the criteria used
to transpose observed associations into a causal relation (Susser, 1986). As already stated and
explained, it was likely due to confounding (Thrusfield, 1997). Given that only one group of
piglets was investigated within each herd, a sufficient number of piglets was tested and also a
large number of herds from different European countries were included. This increases the
validity of the results to other pig herds complying with the selection criteria.
This study showed that, using nested PCR on nasal swabs, M. hyopneumoniae infections in 3weeks old pigs commonly occur in European pig herds suffering from respiratory disease in the
grow-finishing pigs. No significant risk factors directly related to M. hyopneumoniae infections
were found, but the significant variation in within-herd prevalence between herds indicates the
need for further studies aiming to identify risk factors involved in the transmission of M.
hyopneumoniae in suckling pigs. The results also reinforce the need to establish a precise and
detailed diagnosis before implementing a vaccination scheme, as different pathogens may be
involved in respiratory disease.
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CHAPTER 3: EXPERIMENTAL STUDIES
Acknowledgements
The farmers and herd veterinarians are acknowledged for collaboration with this study. Hanne
Vereecke is acknowledged for excellent help in performing the laboratory work.
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CHAPTER 3: EXPERIMENTAL STUDIES
5. References
Grosse Beilage, E., Rohde, N., Krieter, J., 2009. Seroprevalence and risk factors
associated with seropositivity in sows from 67 herds in north-west Germany infected
with Mycoplasma hyopneumoniae. Prev. Vet. Med. 88, 255-263.
Calsamiglia, M., Pijoan, C., 2000. Colonisation state and colostral immunity to
Mycoplasma hyopneumoniae of different parity sows. Vet. Rec. 146, 530-532.
Calsamiglia, M., Pijoan, C., Trigo, A., 1999. Application of a nested polymerase chain
reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs. Journal of Vet.
Diag. Inv. 11, 246-251.
Clark, L.K., Armstrong, C.H., Freeman, M.J., Scheidt, A.B., Sands-Freeman, L., Knox,
K., 1991. Investigating the transmission of Mycoplasma hyopneumoniae in a swine herd
with enzootic pneumonia. Vet. Med. 86, 543-550.
Fano, E., Pijoan, C., Dee, S., 2005. Mycoplasma hyopneumoniae prevalence at weaning
as a predictor of the groups' subsequent Mycoplasma status. In: Proceedings of Allen D.
Leman Swine Conference, Minnesota, USA, pp. 109-113.
Fano, E., Pijoan, C., Dee, S., 2007. Effect of Mycoplasma hyopneumoniae colonization
at weaning on disease severity in growing pigs. Can. J. Vet. Res. 71, 195-200.
Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F., Meyling, A., 1992. A monoclonal blocking
ELISA detecting antibodies to Mycoplasma hyopneumoniae. Vet. Microbiol. 30, 35-46.
Gebruers, F., Calus, D., Maes, D., Villarreal, I., Pasmans, F., Haesebrouck, F., 2008. A
set of four nested PCRs to detect different strains of Mycoplasma hyopneumoniae in
biological samples. In: Proceedings of the 17th Congress of the International
Organisation for Mycoplasmology, Tianjin, China, pp. 90.
Goodwin, R.F., Pomeroy, A.P., Whittlestone, P., 1965. Production of enzootic
pneumonia with a mycoplasma. Vet. Rec. 77, 1247-1249.
99
CHAPTER 3: EXPERIMENTAL STUDIES
Moorkamp, L., Hewicker-Trautwein, M., Grosse, B.E., 2009. Occurrence of
Mycoplasma hyopneumoniae in coughing piglets (3-6 weeks of age) from 50 herds with
a history of endemic respiratory disease. Transboundary Emerging Dis. 56, 54-56.
Maes, D., Verbeke, W., Vicca, J., Verdonck, M., De Kruif, A., 2003. Benefit to cost of
vaccination against Mycoplasma hyopneumoniae in pig herds under Belgian market
conditions from 1996 to 2000. Liv. Prod. Sci. 83, 85-93.
Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M., Haesebrouck, F., 2008. Control
of Mycoplasma hyopneumoniae infections in pigs. Vet. Microbiol. 26, 297-309.
Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F., De Kruif, A., 2004.
Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using
transmission experiments. Prev. Vet. Med. 66, 265-275.
Meyns, T., Maes, D., Calus, D., Ribbens, S., Dewulf, J., Chiers, K., De Kruif, A., Cox,
E., Decostere, A., Haesebrouck, F., 2007. Interactions of highly and low virulent
Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs Vet. Microbiol.
20, 87-95.
Otagiri, Y., Asai, T., Okada, M., Uto, T., Yazawa, S., Hirai, H., Shibata, I., Sato, S.,
2005. Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from
pigs by nested PCR and culture methods. J. Vet. Med. Sci. 67, 801-805.
Ruiz, A.R., Utrera, V., Pijoan, C., 2003. Effect of Mycoplasma hyopneumoniae sow
vaccination on piglet colonization at weaning. Journal of Swine Health Prod. 11, 131135.
Sibila, M., Calsamiglia, M., Vidal, D., Badiella, L., Aldaz, A., Jensen, J.C., 2004.
Dynamics of Mycoplasma hyopneumoniae infection in 12 farms with different
production systems. Can. J. Vet. Res. 68, 12-18.
Sibila, M., Nofrarias, M., Lopez-Soria S., 2007. Exploratory field study on Mycoplasma
hyopneumoniae infection in suckling pigs. Vet. Microbiol. 121, 352-356.
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CHAPTER 3: EXPERIMENTAL STUDIES
Sibila, M., Bernal, R., Torrents, D., Riera, P., Llopart, D., Calsamiglia, M., Segales, J.,
2008. Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet
colonization and seroconversion, and pig lung lesions at slaughter. Vet. Microbiol. 127,
165-170.
Susser, M., 1986. The logic of Sir Karl Popper and the practice of epidemiology. Am. J.
Epidemiol. 124, 711-718.
Thacker, E.L., Thacker BJ, Boettcher TB, Jayappa H., 1998. Comparison of antibody
production, lymphocyte stimulation, and protection induced by four commercial
Mycoplasma hyopneumoniae bacterins. Swine Health Prod. 6, 107-112.
Thacker, E.L, Thanawongnuwech R., 2002. Porcine respiratory disease complex
(PRDC). Thai J. Vet. Med. 32, 125-134.
Thacker, E.L., 2004. Diagnosis of Mycoplasma hyopneumoniae. Animal Health Res.
Rev. 5, 317-320.
Thrusfield, M., 1997. Veterinary Epidemiology. Blackwell Science/University Press,
Cambridge, UK, p. 483.
Villarreal, I., Maes, D., Meyns, T., Gebruers, F., Calus, D., Pasmans, F., Haesebrouck,
F., 2009. Infection with a low virulent Mycoplasma hyopneumoniae isolate does not
protect piglets against subsequent infection with a highly virulent M. hyopneumoniae
isolate. Vaccine 27, 1875-1879.
Zielinski, G., Ross, R.F., 1993. Adherence of Mycoplasma hyopneumoniae to porcine
ciliated respiratory tract cells. Am. J. Vet. Res. 54, 1262-1269.
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102
CHAPTER 3: EXPERIMENTAL STUDIES
3.2 Effect of vaccination against Mycoplasma hyopneumoniae on the
transmission of M. hyopneumoniae under field conditions
I. Villarreal a, *, T. Meyns b, J. Dewulf a, K. Vranckx b, D. Calus b, F. Pasmans b, F. Haesebrouck
b
, D. Maes a
a
Department of Reproduction, Obstetrics and Herd Health and b Department of Pathology,
Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University,
Salisburylaan 133, B-9820 Merelbeke, Belgium
The Veterinary Journal, Article in Press
103
CHAPTER 3: EXPERIMENTAL STUDIES
Abstract
This study investigated the effect of vaccination against Mycoplasma hyopneumoniae on its
transmission in nursery pigs under field conditions. Seventy two pigs were randomly allocated at
weaning into vaccinated (V) and non-vaccinated (NV) groups. Animals in the V group were
vaccinated at 3 weeks-of-age with a commercial M. hyopneumonaie bacterin vaccine.
Bronchoalveolar lavage fluid taken at weaning and at the end of the nursery period was assessed
for the presence of M. hyopneumoniae by nested PCR, and the reproduction ratio of infection
(Rn) was calculated. The percentage of positive pigs in the V and NV groups was 14% and 36%
at weaning, and 31% and 64% at the end of the nursery period, respectively. The Rn-values for
the V and NV groups were 0.71 and 0.56, respectively (P > 0.05). The study indicates that
vaccination does not significantly reduce the transmission of this respiratory pathogen.
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CHAPTER 3: EXPERIMENTAL STUDIES
1. Introduction
Mycoplasma hyopneumoniae is the primary pathogen associated with enzootic pneumonia (EP)
in pigs, a disease that causes significant economic loss to pig production worldwide (Thacker et
al., 1999; Maes et al., 2003). M. hyopneumoniae is mainly transmitted through nose-to-nose
contact, and indirectly via aerosol between susceptible pigs or through airborne transmission
between farms (Goodwin, 1984, 1985; Thomson et al., 1992)
A sound understanding of the epidemiology and especially the transmission of the infection is a
pre-requisite to optimal disease control. The transmission of this pathogen under experimental
conditions has been quantified using a transmission ratio (Rn) (Meyns et al., 2006), and the
values obtained indicated that vaccination did not significantly reduce pathogen transmission
(Meyns et al., 2006). Similar studies quantifying transmission under ‗field‘ conditions have not
been carried out to the authors‘ knowledge but are essential as transmission of infection under
natural conditions may differ significantly from experimental settings. The nursery period in pig
production is central to the transmission of M. hyopneumoniae (Clark et al., 1991; Sibila et al.,
2009). At weaning, pigs infected during the suckling period are transferred to nursery units and
mixed with susceptible non-infected animals, a process that facilitates the spread of pathogens
such as M. hyopneumoniae.
Control of M. hyopneumoniae infection is largely achieved by improving management and
production practices, by the use of antimicrobials and by vaccination (Maes et al., 2008).
Vaccination with the commercially available bacterin vaccines is widely practiced and these
vaccines are efficacious in reducing clinical signs, secondary bacterial respiratory infections, the
105
CHAPTER 3: EXPERIMENTAL STUDIES
extent of lung lesions, and in improving production criteria (Thacker et al., 2004). However,
experimental studies have shown that neither medication nor vaccination prevent colonisation of
the respiratory tract by M. hyopneumoniae (Le Grand and Kobisch, 1996). As infection with this
pathogen under natural conditions commonly occurs in weaned piglets (Sibila et al., 2009), it is
important to assess if vaccination of suckling animals influences the extent of transmission of M.
hyopneumoniae during the nursery period. The aim of this study was to quantify the transmission
of M. hyopneumoniae in nursery pigs under field conditions, and to assess the effect of
vaccination on this parameter. In addition, animal production criteria and the extent of lung
lesions at slaughter were assessed.
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CHAPTER 3: EXPERIMENTAL STUDIES
2. Materials and methods
Details of herd under study
The study was conducted in 2008 in a Belgian pig herd comprising 1200 commercial hybrid
sows (PIC) that operated a two-week batch production system. The sows were inseminated
Piétrain semen. The reproductive performance of the sows (average number of live-born piglets,
11), and piglet health were good, although there had been some disease due to Streptococcus suis
infection. The pre-weaning and nursery period mortality rates were approximately 9% and 2%,
respectively. Piglets were cross-fostered during the first 3 days after birth and were weaned at 21
days-of-age. The piglets were given an iron injection at 2 days of age and were medicated with
ceftiofur (Naxcel, Pfizer Animal Health) at 5 days-of-age in response to the S. suis infection.
Limited sampling using nasal swabs prior to the study indicated that 8/20 3 week-old piglets and
20/20 7 week-old piglets were positive for M. hyopneumoniae by nested (nPCR). No clinical
signs of respiratory disease were evident in nursery piglets. The sows were vaccinated against M.
hyopneumoniae (Porcilis Mhyo, Intervet Schering-Plough Animal Health) at 6 and 4 weeks prior
to farrowing.
Experimental design
In total, 72 piglets were selected at weaning for study. Following standard management practice
in the herd, all piglets were mixed after weaning and grouped into three weight categories (low,
moderate and high weaning weight), based on visual assessment. The pigs used in the study were
then randomly selected from the moderate weaning weight group. These animals were
individually identified by ear-tag number, and randomly assigned to a vaccinated (V) (n = 36) or
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CHAPTER 3: EXPERIMENTAL STUDIES
non-vaccinated (NV) (n = 36) group. Pigs of the V group were vaccinated IM at 3 weeks-of-age
with an inactivated M. hyopneumoniae vaccine (Suvaxyn MH-One, Fort Dodge Animal Health).
Pigs in the NV group were left untreated. Both groups were housed in separate nursery
accommodation for 6 weeks after weaning. In each group all 36 animals were housed in one pen
with solid partitions to eliminate nose-to-nose contact with other pigs in the same nursery room.
The stocking density was 0.26 m²/pig, the flooring was fully slatted, and the piglets were fed ad
libitum with a commercial feed.
Each nursery room contained 16 pens (8 on each side of a central corridor) with approximately
36 pigs/pen. In consequence, the capacity of each nursery room was approximately 576 animals.
In the nursery room containing the NV group, the pigs in the adjacent pens not involved in the
trial were left unvaccinated, whereas those in the nursery room with the V group were vaccinated
at 3 weeks-of-age. The rooms were mechanically ventilated via ceiling air inlets. At the end of
the nursery period, the piglets were moved to a fattening unit located 1 km away. Here, the pigs
under study were housed in four different pens in the same room with a capacity for
approximately 144 pigs until they reached slaughter age (27 weeks). The pigs from the V group
in the adjacent pens, were vaccinated at the start of the fattening period (Porcilis Mhyo, Intervet
Schering-Plough Animal Health). The stocking density in the fattening unit was 0.77 m²/pig.
Broncho-alveolar lavage sampling, nasal swabbing, nPCR analysis and bacteriological
examination
All 72 piglets were anaesthesised with 0.22 mL/kg of a mixture of xylazin (Xyl-M 2%, VMD)
and zolazepam and tiletamin (Zoletil 100, Virbac) at 3 and 9 weeks-of-age to collect broncho108
CHAPTER 3: EXPERIMENTAL STUDIES
alveolar lavage (BAL) fluid. Catheters with a diameter of 3.3 mm were used and 10 mL of
phosphate buffered saline (PBS) was infused into the lungs and the fluid then gently aspirated.
At 27 weeks-of-age, the lungs were removed at slaughter and immediately transported at 4 °C to
the Faculty of Veterinary Medicine at Gent University. BAL was carried out on each right lung
as described above except using 20 mL of PBS. The aspirated fluid was centrifuged at 4000 g for
30 min and the resulting pellet re-suspended in 1 mL of PBS and stored at –80 °C until DNA
extraction was performed. DNA was extracted using a DNeasy blood and tissue kit (Qiagen), and
a nPCR test was performed on the extract (Stärk et al., 1998). An animal was considered infected
if the BAL fluid was found positive by nPCR.
Nasal swabs were collected at 3, 9 and 27 weeks-of-age from all animals. DNA was extracted
with the DNeasy kit and nPCR was performed on the extract. A sample of each BAL fluid was
inoculated onto Columbia and Columbia CNA agar each supplemented with 5% sheep blood
(Oxoid). The Columbia agar also contained a Staphylococcus intermedius streak to support
growth of Actinobacillus and Haemophilus spp.. Plates were incubated overnight in a 5% CO2enriched environment at 37 °C and isolated bacteria were identified (Quinn et al., 1994).
Serological examination
Blood samples were taken at 3 and 9 weeks-of-age, and at slaughter. Serological examination to
detect antibodies against M. hyopneumoniae, was carried using an ELISA (DAKO Mh) (Feld et
al., 1992). Sera with optical density (OD) values < 50% of the ODbuffer-control were considered
positive. OD values ≥ 50% of the ODbuffer-control were classified as negative. Blood samples were
also tested for antibodies against: porcine reproductive and respiratory disease virus (PRRSV)
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CHAPTER 3: EXPERIMENTAL STUDIES
(HerdChek PRRS, IDEXX); porcine circovirus type-2 (PCV2) (PCV2 ELISA, Shenzhen
Lvshiyuan Biotechnology Co.); and porcine influenza sub-type H1N1 (HerdChek Swine
Influenza virus, IDEXX).
Assessment of weight gain and of lung lesions
Pigs in the V and NV groups were individually weighed at 3, 9 and 26 weeks-of-age to
determine average daily weight gain (ADG). ADG for these periods was estimated from the
difference between the starting and final weight, divided by the number of days during that
period. At slaughter, macroscopically visible pneumonia was quantified in a blinded fashion
(Hannan et al., 1982). The scoring ranged from ‗0‘ (no lesions) to 35 (entire lung affected). A
Slaughterhouse Pleurisy Evaluation System (SPES) was used to grade any attendant pleurisy
(Ostanello et al., 2007) and scores ranged from ‗0‘ to ‗4‘.
Statistical analysis
Estimation of the degree of inter-animal transmission of M. hyopneumoniae during the nursery
period (when pigs were between 3 and 9 weeks-of-age) in each group was carried out using a
stochastic infection model and by means of an adjusted reproduction ratio (Rn) (Meyns et al.,
2004). In brief, this model assumes that the process of transmission between piglets is in
accordance with the susceptible–infectious (S–I) model. This model was selected since it
assumes that once an animal is infected, it does not recover before the end of the trial. At the
start of the experiment, the number of M. hyopneumoniae-positive BAL fluid samples was
determined by means of nPCR. The number of new contact infections was then determined by
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CHAPTER 3: EXPERIMENTAL STUDIES
the number of positive BAL fluid samples at the end of the trial, which in turn determines the
scale of the outbreak. Based on this final number of infected animals, the number of infectious
pigs at weaning (the start of the study), the transmission ratio can be estimated using the
algorithm described by De Jong and Kimman (1994). This ratio is defined as the mean number
of secondary cases caused by one infectious pig during the nursery period. The calculation of
transmission ratio takes into account the different numbers of infected pigs in the V and NV
groups at the start of the study (Kluivers et al., 2006). In this context, the different number of
nPCR-positive animals at 3 weeks-of-age did not biase the Rn values.
The prevalence of pigs seropositive for M. hyopneumoniae in the V and NV groups was
analysed using logistic regression. Pig weights were compared between groups using a
Student‘s t test for independent samples. Lung lesion scores were compared using a nonparametric Wilcoxon test. Result were considered statistically significant when P < 0.05 and
data were analysed using SPSS 16.0 (SPSS Inc.).
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CHAPTER 3: EXPERIMENTAL STUDIES
3. Results
Detection of M. hyopneumoniae by nPCR in broncho-alveolar lavage fluid
The number of nPCR-positive BAL fluid samples at 3, 9 and 27 weeks-of-age are presented in
Table 1. The percentage of positive samples at 3 weeks was 14% (5/36) and 36% (13/36) in the
V and NV groups, respectively (P > 0.05). At 9 weeks of age, the percentage of positive
samples in the V and NV groups was 31% (11/36) and 64% (23/36), respectively (P < 0.05). All
pigs that tested positive at weaning remained positive at 9 weeks-of-age.
Of the nPCR-negative BAL fluid samples at 3 weeks-of-age (n = 31, V group; n = 23, NV
group), 19% (6/31) were in the V group and 43% (10/23) of the NV group became positive at 9
weeks-of-age (P = 0.05). It follows that six and ten animals developed infection during the
nursery period in the V and NV groups, respectively. The Rn values (95% confidence interval)
for the V and NV groups during the nursery period were estimated as Rn= 0.71 (0.32, 2.06) and
Rn= 0.56 (0.29, 1.05), respectively (P > 0.05). For practical reasons, it was not possible to
sample all the study pigs at slaughter so that 32 and 26 animals were sampled from the V and
NV groups, respectively. The percentage of nPCR-positive pigs at slaughter was 59% and 7% in
these groups, respectively (P < 0.05).
Detection of M. hyopneumoniae by nPCR in nasal swabs
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All swabs from pigs at 3 weeks-of-age were negative on nPCR (Table 1). At 9 weeks-of-age,
the percentage of nPCR-positive animals was 31% and 58% in the V and NV groups,
respectively (P < 0.05). Nasal swabbing at slaughter was not carried out as such a procedure
was likely to be adversely affected by carcase treatment procedures (Marois et al., 2008).
Detection of M. hyopneumoniae by bacteriological culture of broncho-alveolar lavage fluid
S. suis was the bacterium most frequently isolated from BAL fluid samples from both groups
(Table 1). At 9 weeks-of-age, the prevalence of this pathogen was significantly lower in the V
than in the NV group (P < 0.05). Staphylococcus aureus was isolated from BAL fluid samples
in only one pig in the NV group at 3 weeks-of-age and at slaughter. Actinobacillus
pleuropneumoniae was isolated from only one piglet in the V group at 3 weeks-of-age.
Table 1: Results of the nested PCR (nPCR) assessment of broncho-alveolar lavage (BAL) fluid
and nasal swab samples, and of bacteriological examination of BAL fluid in pigs vaccinated (V)
and not vaccinated (NV) against Mycoplasma hyopneumoniae at 3 weeks-of-age (weaning), 9
weeks-of-age (end of nursery phase of production) and at 27 weeks-of-age (slaughter). In total,
72 pigs from one herd were studied.
113
Age of pigs at sampling
3 weeks
9 weeks
27 weeks
(weaning)
(end of nursery period)
(slaughter) a
V
NV
P
V
NV
value
Number (%) of nPCRpositive pigs on BAL
fluid
5/36
13/36 (36)
Number (%) of nPCRpositive pigs on nasal
swabs
0/36
0/36
(0)
(0)
6/36 (17)
8/36 (22)
0/36 (0)
0/36 (0)
0.05
(14)
V
NV
P
value
19/32
(59)
2/26
<0.01
0.02
-
-
-
value
11/36
(31)
6 newly
infected
>0.05
P
23/36 (64)
<0.01
10 newly
infected
(7)
11/36
(31)
21/36
0.38
0/36 (0)
6/36 (17)
0.01
7/32 (22)
5/26 (19)
0.53
1/36 (3)
0.50
0/36 (0)
0/36 (0)
>0.05
0/32 (0)
1/26 (4)
0.45
1/36 (3)
0.50
0/36 (0)
0/36 (0)
>0.05
0/32 (0)
0/26 (0)
>0.05
(58)
Number (%) of pigs
positive
for
the
following pathogens on
bacteriological culture
of BAL fluid:
Streptococcus suis
Staphylococcus aureus
b
A. pleuropneumoniae
a
Not all pigs could be sampled at slaughter for practical reasons and nasal swabs were not taken at slaughter. b A. pleuropneumoniae,
Actinobacillus pleuropneumoniae.
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3: EXPERIMENTAL STUDIES
Serological examinations
The percentage of pigs seropositive for M. hyopneumoniae at 3 weeks-of-age was 33% and 14%
in the V and NV groups, respectively (P > 0.05) (Table 2). At 9 weeks-of-age, the percentage of
seropositive pigs in the V and NV groups was 42% and 3%, respectively (P < 0.05). At
slaughter, the percentage of seropositive pigs in the V and NV groups was 56% and 35% (P >
0.05), respectively. The average serum OD values (SD) for M. hyopneumoniae in the V and
NV groups are detailed in Table 2. The percentage of pigs seropositive for PRRSV, PCV2 and
H1N1 at 3, 9 and 27 weeks-of-age were not significantly different between the two groups (P >
0.05) (Table 2).
Table 2: Serological assessment and weights of pigs vaccinated (V) and not vaccinated (NV)
against Mycoplasma hyopneumoniae at 3 weeks-of-age (weaning), 9 weeks-of-age (end of
nursery phase of production) and at 26 weeks-of-age (one week prior to slaughter). In total, 72
pigs from one herd were studied. Animals were assessed for antibodies to M. hyopneumoniae,
porcine reproductive and respiratory disease virus (PRRSV), porcine circovirus type-2 (PCV2),
and porcine influenza sub-type H1N1.
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3: EXPERIMENTAL STUDIES
Age of pigs at sampling
3 weeks
9 weeks
27
(weaning)
(end of nursery period)
(slaughter) a
V
V
V
NV
P
NV
value
12/36
10/36
(33)
(14)
69.4
74.6
(47.3)
(32.9)
Number (%) of pigs
seropositive for PRRSV
15/36
18/36
(41)
(50)
Number (%) of pigs
seropositive for PCV2
25/36
23/36
(69)
(64)
Number (%) of pigs
seropositive for porcine
influenza (H1N1)
34/36
31/36
(94)
(86)
Average weight (± SD)
(kg)
7.4
7.4
(1.0)
(0.9)
Number (%) of pigs
seropositive for M.
hyopneumoniae
Average OD
sera for
b
value of
0.40
0.67
P
NV
value
15/36
1/36
(42)
(3)
53.0
77.9
(24.3)
(14.5)
28/36
29/36
(78)
(81)
22/36
19/36
(61)
(53)
20/36
22/36
(56)
(61)
15.9
17.6
(2.9)
(2.9)
<0.01
<0.01
P
value
14/25
7/20
(56)
(35)
45.8
65.5
(20.1)
(18.8)
14/25
16/20
(56)
(80)
10/25
13/20
(40)
(65)
13/25
8/20
(52)
(40)
117.9
112.8
0.01
0.81
M. hyopneumoniae (±
SD)
0.32
0.40
0.21
0.87
0.04
0.32
0.41
0.32
(2.0)
(34 pigs)
a
0.08
0.08
0.31
0.21
(3.4)
c
(35 pigs)
Not all pigs could be sampled at slaughter for practical reasons. b OD, optical density.c Fattening pigs were weighted at 26 weeksof-age, one week prior to slaughter.
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3: EXPERIMENTAL STUDIES
Average daily weight gain and extent of lung lesions
The weight of the pigs at 3, 9 and 27 weeks-of-age in the V and NV groups are detailed in
Table 2. On average, pigs of the V group gained 5.1 kg more than pigs in the NV group
between 3 and 26 weeks-of-age (P > 0.05). The ADG was significantly lower during the
nursery period in the V group than in the NV group, whereas the opposite pertained during
the fattening period (week 9 to 26) (Table 3). Over the entire period of the study, ADG was
higher in the V than in the NV group (581.9 ± 55.7 g/day versus 555.0 ± 102.6 g/day),
although not significantly so (P > 0.05) (Table 3).
Table 3: Differences in average daily weight gain (ADG) (mean  SD) between pigs
vaccinated (V) and not vaccinated (NV) against Mycoplasma hyopneumoniae during the
nursery period (3 to 9 weeks-of-age), the fattening period (9 to 26 weeks-of-age), and over the
entire study (3 to 26 weeks-of-age). In total, 72 pigs from one herd were studied.
Age range of ADG (g)
(weeks)
Difference in Difference
ADG (V-NV ADG
animals) (g) (%)
V
in P
value
NV
3 to 9
217.3
54.9
±
9 to 26
667.1 ± 6.1 617.9 ± 2.7
3 to 26
581.9
55.7
±
285.9 ± 57.9
-70.7
-24.0
<0.01
+49.2
+7.9
<0.01
555.0 ± 102.6 +29.9
+4.8
0.19
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3: EXPERIMENTAL STUDIES
The percentage of pigs with pneumonic lesions in the V and NV groups was 81 and 88%,
respectively (P > 0.05) (Table 4). The average lesion scores in the V and NV groups was
0.97 and 0.71, respectively (P > 0.05). The average SPES score was 1.37 ± 1.7 for the V
group and 0.67 ± 0.9 for the NV group, respectively (P < 0.05) (Table 4).
Table 4: Details of the extent of pneumonic lesions and pleuritis in pigs vaccinated (V) and
not vaccinated (NV) against Mycoplasma hyopneumoniae at slaughter at 27 weeks-of-age. In
total, 72 pigs from one herd were studied.
Parameter
Group
P
value
V
NV
Number (%) of pigs with pneumonia
26/32 (81)
23/26 (88)
0.22
Average (± SD) pneumonia score
0.97 (1.5)
0.71 (0.9)
0.46
Number (%) of pigs with pleuritis
17/32 (53)
16/26 (62)
0.35
Average (± SD) pleuritis score
1.37 (1.7)
0.67 (0.9)
0.04
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3: EXPERIMENTAL STUDIES
4. Discussion
This study has quantified the transmission of M. hyopneumoniae in nursery-phase pigs under
field conditions, and has assessed the effect of single-dose vaccination on pathogen
transmission. The work additionally investigated the prevalence of M. hyopneumoniae
infection, animal performance during fattening and the severity of lung lesions at slaughter.
The calculated Rn-values indicate that, during the six week nursery period, infected pigs infect
on average 0.56 susceptible pen-mates. This value is lower than that previously found in
transmission studies under experimental conditions (Meyns et al., 2004, 2006), and may be
explained by factors such as stocking density, animal immune status and infection dose. The
dose used to inoculate pigs in previous experimental studies (Meyns et al., 2004, 2006) was
relatively high. Although the infecting dose under field conditions is not know, it would be
expected to be lower, especially where good herd management and housing prevail, as in the
present study. A lower infection dose is likely to result in lower numbers of organisms in the
respiratory tract and thus a lower transmission rate and Rn value.
Similar to previous experimental transmission studies (Meyns et al., 2004, 2006), the current
work indicates that vaccination does not significantly reduce M. hyopneumoniae transmission
under field conditions and suggests that vaccination alone will not eliminate infection with
this pathogen from herds. The lack of any vaccination effect on transmission may have been
due to the considerable numbers of pigs already infected at the time of vaccination at 3
weeks-of-age (14% in V and 36% in NV groups, respectively). The finding that pigs positive
at vaccination remained positive at 9 weeks-of-age was not unexpected as vaccination of
infected animals was not likely to result in the rapid clearance of M. hyopneumoniae from the
lungs (Maes et al., 2008).
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3: EXPERIMENTAL STUDIES
The 25% overall prevalence of infection at weaning in BAL fluid was higher than previously
reported (Vicca et al., 2002; Sibila et al., 2007), although previous studies only carried out
nPCR-detection on nasal swabs. Our results support those of a previous study that found more
infected pigs on analysis of BAL fluid samples than of nasal swabs (Kurth et al., 2002), a
finding particularly true of the 3 week-old piglets in our study. We chose to calculate the
transmission ratio from the nPCR results from the BAL fluid and not from the nasal swabs, as
M. hyopneumoniae is primarily found adherent to the ciliated epithelium of the trachea,
bronchi and bronchioles rather than in the nasal chambers (Blanchard et al., 1992).
Furthermore, although the assessment of nasal swabs may be suitable for investigating
infection prevalence at a herd level, it is not appropriate in assessing the infection status of
individual animals (Sibila et al., 2009). Nasal swabs were not taken at slaughter as
contaminated water used in carcase treatment can reach the lungs, leading to the possibility of
false positive results (Marois et al., 2008)
Given the high prevalence (88%) of lung lesions in the NV group at slaughter, it was
surprising to find a low percentage of nPCR-positive results (7%) on analysis of BAL fluid
from this group. Although the explanation for this remains unclear it is possible that the lungs
of the NV animals contained greater amounts of DNA from other bacteria or from the blood
within the lungs at slaughter. This DNA may not have been degraded fully during the
extraction procedure and could have acted as an amplification inhibitor during nPCR
(Baumeister et al., 1998). nPCR on BAL fluid from which the DNA is extracted using the
QIAamp blood kit can be less sensitive in detecting M. hyopneumoniae when amplification
inhibitors are not fully removed (I. Villarreal, personal communication).
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3: EXPERIMENTAL STUDIES
The finding of a high prevalence of infected pigs at 3 weeks-of-age was somewhat surprising
as the sows were vaccinated twice before farrowing. Vaccinating sows 5 and 3 weeks prior to
farrowing induces high maternal antibody titres in suckling piglets and it is associated with
lower numbers of infected animals at weaning as detected by nPCR on nasal swabs (Ruiz et
al., 2003; Sibila et al., 2007, 2008a). The higher number of seropositive piglets at 3 weeks-ofage, compared to 9 weeks-of-age, was due to the effect of maternal antibody. At 3 weeks-ofage, the percentage of seropositive pigs was higher in the V (33%) than in the NV (14%)
group and the average OD values were slightly lower (69.4 vs. 74.6). It remains unknown if
these differences could have accounted for the lower infection prevalence found by nPCR on
BAL fluid samples from the V group at this age. Maternally-derived antibodies, under
experimental conditions, provide limited to no effect on respiratory tract colonisation by M.
hyopneumoniae (Thacker et al., 2000). Since our transmission ratios take into account the
numbers of infected pigs in both groups at the start of the study, the different numbers of
nPCR-positive animals at 3 weeks-of-age have not biased these calculations.
The influence of maternal antibody on the vaccine response in the present study is not clear.
As the efficacy of vaccination of suckling pigs has been demonstrated under both
experimental and field conditions (Jensen et al., 2002), it is unlikely that such antibodies
significantly influenced the host response to vaccination. Antibody levels can remain the
same (Martelli et al., 2006), or be reduced (Maes et al., 1999; Hodgins et al., 2004) following
the vaccination of pigs with high levels of maternally-derived antibody. Thus as the optimal
timing of vaccination must balance the advantages of delayed vaccination with the need to
induce adequate early immunity, and given the considerable number of infected pigs we
found at weaning, the early vaccination strategy we adopted in this study appears justified.
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3: EXPERIMENTAL STUDIES
The reason for the significantly lower ADG during the nursery phase of production in the V
than in the NV group is not clear. However, during the fattening phase and by 26 weeks-ofage, the ADG was significantly higher in the V than in the NV pigs. This finding is in
agreement with previous studies (Jensen et al., 2002). Although studies show that a singledose vaccination protocol can decrease the incidence of clinical disease and of lung lesions at
slaughter (Dawson et al., 2002; Moreau et al., 2004), in the present study no significant
difference in the percentage of pigs with pneumonic lesions or in the severity of these lesions
was found between the groups. The reason for this finding is not known, but it may be due
to: the small study population; the effects of re-infection; the fact that vaccinated and nonvaccinated animals were housed in the same accommodation during the fattening period; or
the absence of significant clinical disease in this case. Our findings are consistent with those
of a previous study (Vicca et al., 2002), where a high prevalence of infection, even at a
young age, did not necessarily correlate with a higher incidence of clinical disease or with
more substantial lung lesions at slaughter. The lack of clinical disease experienced in our
study herd may be attributed to the good management and housing conditions pertaining, and
also to the fact that the strain of M. hyopneumoniae circulating in this herd was of low
virulence (Vicca et al., 2003).
This field study has found that, similar to previous transmission studies of M. hyopneumoniae
under experimental conditions (Meyns et al., 2006), vaccination with a bacterin-based vaccine
does not significantly reduce the transmission of this pathogen between pigs. This finding
implies that vaccination alone will not be effective in eliminating M. hyopneumoniae
infection from herds.
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Acknowledgements
This research was financially supported by Fort Dodge Animal Health. The authors are
grateful to the herd-owner for their collaboration as well as to Hanne Vereecke for laboratory
support.
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3: EXPERIMENTAL STUDIES
5. References
Baumeister, K., Runge, M., Ganter, M., Feenstra, A., Delbeck, F., Kirschhoff, O., 1998.
Detection of Mycoplasma hyopneumoniae in bronchoalveolar lavage fluids of pigs by PCR. J.
Clin. Microbiol. 36, 1984-1988.
Blanchard, B., Vena, M.M., Cavalier, A., Le, L.J., Gouranton, J., Kobisch, M. 1992. Electron
microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma
hyopneumoniae. Vet. Microbiol. 30, 329-341.
Clark, L.K., Armstrong C. H., Freeman, M. J., Scheidt, A.B., Sands-Freeman, L., Knox, K.,
1991. Investigating the transmission of Mycoplasma hyopneumoniae in a swine herd with
enzootic pneumonia. Vet. Med. 86, 543-550.
Dawson, A., Harvey, R.E., Thevasagayam, S.J., Sherington, J., Peters, A.R., 2002. Studies of
the field efficacy and safety of a single-dose Mycoplasma hyopneumoniae vaccine for pigs.
Vet. Rec. 151, 535-538.
De Jong, M., Kimman, T., 1994. Experimental quantification of vaccine-induced reduction in
virus transmission. Vaccine 100, 255-268.
Feld, N., Quist, P., Ahrens, P., Friis, N. and Meyling, A., 1992. A monoclonal blocking
ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. Vet. Microbiol. 30, 35–
46.
Goodwin, R.F., 1984. Apparent reinfection of enzootic-pneumonia-free pig herds: early signs
and incubation period. Vet. Rec. 115, 320-324.
Goodwin, R.F., 1985. Apparent reinfection of enzootic-pneumonia-free pig herds: search for
possible causes. Vet. Rec. 116, 690-694.
Hannan, P.C., Bhogal, B.S., Fish, J.P., 1982. Tylosin tartrate and tiamutilin effects on
experimental piglet pneumonia induced with pneumonic pig lung homogenate containing
mycoplasmas, bacteria and viruses. Res. Vet. Sci. 33, 76-88.
Hodgins, D., Shewen, P., Dewey, C., 2004. Influence of age and maternal antibodies on
antibody responses of neonatal piglets vaccinated against Mycoplasma hyopneumoniae. J.
Swine Health Prod. 12, 10-16.
124
3: EXPERIMENTAL STUDIES
Jensen, D., Ersboll, A., Nielsen, J., 2002. A meta-analysis comparing the effect of vaccines
against Mycoplasma hyopneumoniae on daily weight gain in pigs. Prev. Vet. Med. 54, 265278.
Kluivers, M., Maurice, H., Vyt, P., Koenen, F., Nielen, M., 2006. Transmission of
encephalomyocarditis virus in pigs estimated from field data in Belgium by means of R0. Vet.
Res. 37, 757-766.
Kurth, K., Hsu, T., Snook E., Thacker E., Thacker B., Minion C., 2002. Use of a Mycoplasma
hyoneumoniae nested polymerase chain reaction test to determine the optimal sampling sites
in swine. J. Vet. Diag. Inv. 14, 463-469.
Le Grand, A., Kobisch, M., 1996. Comparison of the use of a vaccine and sequential
antibiotic treatment in a herd infected with Mycoplasma hyopneumoniae. Vet. Res. 27, 241253.
Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., Verbeke, W.,
Viaene, J., de Kruif, A., 1999. Effect of vaccination against Mycoplasma hyopneumoniae in
pig herds with an all-in/all-out production system. Vaccine 17, 1024-1034.
Maes, D., Verbeke, W., Vicca, J., Verdonck, M., De Kruif, A., 2003. Benefit to cost of
vaccination against Mycoplasma hyopneumoniae in pig herds under Belgian market
conditions from 1996 to 2000. Livest. Prod. Sci. 83, 85-93.
Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M., Haesebrouck, F., 2008. Control of
Mycoplasma hyopneumoniae infections in pigs. Vet. Microbiol. 126, 297-309.
Marois, C., Cariolet, R., Morvan, H., Kobisch, M., 2008. Transmission of pathogenic
respiratory bacteria to specific pathogen free pigs at slaughter. Vet. Microbiol. 129, 325-332.
Martelli, P., Terreni, M., Guazzetti, S., Cavirani, S., 2006. Antibody response to Mycoplasma
hyopneumoniae infection in vaccinated pigs with or without maternal antibodies induced by
sow vaccination. J. Vet. Med. Sci. B 53, 229-233.
Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F., de, K.A., 2004. Quantification of
the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments.
Prev. Vet. Med. 66, 265-275.
125
3: EXPERIMENTAL STUDIES
Meyns, T., Dewulf, J., de, K. A., Calus, D., Haesebrouck, F., Maes, D., 2006. Comparison of
transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations.
Vaccine 24, 7081-7086.
Moreau, I.A., Miller, G.Y., Bahnson, P.B., 2004. Effects of Mycoplasma hyopneumoniae
vaccine on pigs naturally infected with M. hyopneumoniae and porcine reproductive and
respiratory syndrome virus. Vaccine 22, 2328-2333.
Ostanello, F., Dottori, M., Gusmara, C., Leotti, G., Sala, V., 2007. Pneumonia disease
assessment using a slaughterhouse lung-scoring method, J. Vet. Med. 54, 70–75.
Quinn, P.J., Carter, M., Markey, B., Carter, G., 1994. In: Clinical Vet. Microbiol.. Mosby,
Edinburgh, UK, pp. 254-259.
Ruiz, A., Utrera, V., Pijoan, C., 2003. Effect of Mycoplasma hyopneumoniae sow vaccination
on piglet colonization at weaning, J. Swine Health Prod. 11, 131–135.
Sibila, M., Nofrarias, M., Lopez-Soria, S., Segales, J., Riera, P., Llopart, D., Calsamiglia, M.,
2007. Exploratory field study on Mycoplasma hyopneumoniae infection in suckling pigs. Vet.
Microbiol. 121, 352-356.
Sibila, M., Bernal, R., Torrents, D., Riera, P., Llopart, D., Calsamiglia, M., Segales, J., 2008a.
Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet
colonization and seroconversion, and pig lung lesions at slaughter. Vet. Microbiol. 127, 165170.
Sibila, M., Pieters, M., Molitor, T., Maes, D., Haesebrouck, F., Segales, J., 2009. Current
perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection.
Vet. J. 181, 221-231.
Stark, K.D., Nicolet, J., Frey, J., 1998. Detection of Mycoplasma hyopneumoniae by air
sampling with a nested PCR assay. Appl. Environ. Microbiol. 64, 543-548.
Thacker, E.L., Halbur, P.G., Ross, R.F., Thanawongnuwech, R., Thacker, B.J., 1999.
Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome
virus-induced pneumonia. J. Clin. Microbiol. 37, 620-627.
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3: EXPERIMENTAL STUDIES
Thacker, B., Thacker, E., Halbur, P., Minion, C., Young, T., Erickson, B., Thanawonguwech,
T., 2000. The influence of maternally-derived antibodies on Mycoplasma hyopneumoniae
infection. In: Proceedings of the 16th Conference of the International Pig Veterinary Society,
Melbourne Australia, p. 454.
Thacker, E.L. 2004. Diagnosis of Mycoplasma hyopneumoniae. Ani. Health Res. Rev. 5, 317320.
Thomson, B.L., Jorsal, S.E., Andersen, S., Willeberg, P., 1992. The Cox regression model
applied to risk factor analysis of infections in the breeding and multiplying herd in the Danish
SPF system. Prev. Vet. Med. 12, 287-297.
Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F., de, K.A., 2002. Patterns of
Mycoplasma hyopneumoniae infections in Belgian farrow-to-finish pig herds with diverging
disease-course. J. Vet. Med. B Inf. Dis. Vet. Public Health 49, 349-353.
Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de, K. A., Haesebrouck, F., 2003.
Evaluation of virulence of Mycoplasma hyopneumoniae field isolates. Vet. Microbiol. 97,
177-190.
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128
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Protection against different strains of Mycoplasma
hyopneumoniae
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3.3 Effect of vaccination of pigs against experimental infection with highly
and low virulent Mycoplasma hyopneumoniae strains
I.Villarreala, D. Maesa, K. Vranckxb, D. Calusb, F. Pasmansb, F. Haesebrouckb
a
Department of Reproduction, Obstetrics and Herd Health,
b
Department of Pathology, Bacteriology and Avian Diseases.
Ghent University, Faculty of Veterinary Medicine. Salisburylaan 133, B-9820 Merelbeke,
Belgium.
Vaccine, Submitted Oct, 2010
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Abstract
This study investigated the infection pattern and lung lesion development in pigs caused by a
low and highly virulent M. hyopneumoniae strain at 4 and 8 weeks (w) post infection (PI). It
also determined the efficacy of a commercial inactivated whole-cell vaccine against infection
with each one of these M. hyopneumoniae strains. Ninety piglets free of M. hyopneumoniae
were selected, and 40 of them were randomly vaccinated during their first week of life. At
weaning, all piglets were allocated to 10 different groups and housed in pens with absolute
filters. At 4 weeks of age, pigs were inoculated intratracheally with either a highly virulent M.
hyopneumoniae strain, a low virulent strain or with sterile culture medium. Half of all animals
were euthanized at 4w PI, while the remaining half was euthanized at 8w PI. Coughing was
assessed daily, and lung lesions, immunofluorescence (IF), bacteriological analysis and nested
PCR were assessed after necropsy. It was demonstrated that contrary to the highly virulent
strain, the low virulent strain required more than 4 weeks PI (commonly accepted as the
standard infection model) to reach maximum clinical symptoms. Vaccination significantly
reduced clinical symptoms, macroscopic and microscopic lung lesions in pigs infected with
the highly virulent strain. This effect was more pronounced at 4 than at 8 weeks PI. Protective
efficacy was also observed in pigs infected with the low virulent strain, but the effect was less
pronounced than with the highly virulent strain.
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1. Introduction
Mycoplasma hyopneumoniae (M. hyopneumoniae) is the causative agent of enzootic
pneumonia in swine, a disease with worldwide economic impact in intensive pig production
(Thacker et al., 1999; Maes et al., 2008) and characterized by chronic non productive
coughing, retarded growth rate and poor feed conversion ratio (Ross 1992).
It is well known that factors such as management practices and housing conditions influence
the infection pattern and the severity of the disease in pig herds (Maes et al 1999). However,
also the virulence of M. hyopneumoniae strains has been demonstrated to determine the
clinical course of the infection (Vicca et al., 2003). Experimental infection with a highly
virulent M. hyopneumoniae strain was associated with severe clinical signs and lung lesions 4
weeks post infection, whereas clinical symptoms and lung lesions were milder following
infection with low virulent strains (Meyns et al., 2006; Vicca et al., 2003).
In most M. hyopneumoniae experimental infection studies, pigs are euthanized at 4 weeks
post infection (PI), based on the fact that macroscopic lung lesions reach their maximum size
2-4 weeks after inoculation (Whittlestone et al., 1972; Kobisch et al., 1993). This model has
been widely accepted as the standard infection model for this pathogen. However, recent
experiments showed that, depending on the strain, the most severe clinical symptoms and
lung lesions are sometimes seen later than 4 weeks after infection (Villarreal et al., 2009). As
a result, the standard infection model might not be suitable for studying all types of M.
hyopneumoniae strains, as some of them might require more than 4 weeks PI to fully develop
symptoms and lesions in the infected pigs.
Vaccination is frequently practiced to control M. hyopneumoniae infections in pig herds
worldwide. Although vaccination with the currently available bacterin vaccines is not able to
prevent transmission nor establishment of the microorganism in the lungs (Meyns et al., 2006;
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Thacker et al. 1998), numerous studies have shown that it reduces the clinical symptoms and
lung lesions, improves performance, and leads to an economic benefit for the pig producers
(Maes et al., 2003; Moreau at al., 2004; Maes et al., 2008; Sibila et al., 2007). M.
hyopneumoniae is, however, a highly heterogenous species not only with regard to virulence
(Meyns et al., 2006; Vicca et al., 2003), but also at genetic, antigenic and proteomic level
(Calus et al., 2010; Pinto et al., 2009; Stakenborg et al., 2005). Most of the currently available
bacterin vaccines are based on an adjuvanted suspension of inactivated M. hyopneumoniae
organisms, and it is not clear whether the protective effect of vaccination may vary depending
on the strain causing the infection.
Therefore, the aims of this study were twofold. First, in order to assess the suitability of the
standard infection model, the present study investigated the infection pattern and lung lesion
development in pigs infected by low and highly virulent M. hyopneumoniae strains during 8
weeks after infection. Secondly, the effect of vaccination with a commercial vaccine against
infection with M. hyopneumoniae strains of low and high virulence was evaluated.
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2. Materials and methods
M. hyopneumoniae strains and intratracheal inoculation procedure
For experimental infection of pigs, the low virulent strain (F13.7B) and the highly virulent
strain (F7.2C) were used (Vicca et al., 2003; Meyns et al., 2007). Both strains have been
differentiated at a genomic and proteomic level as described by Stakenborg et al., (2006) and
Calus et al., (2007).
For experimental infection, pigs were anesthesized with 0.22 ml/kg of a mixture of Xyl-M®
2% (VMD, Arendonk, Belgium) and Zoletil 100® (Virbac, Louvain la Neuve, Belgium), and
they were inoculated intratracheally with 7 x 107 CCU/ml of either the low or highly virulent
M. hyopneumoniae strain in 7 ml inoculum, or with 7 ml of sterile culture medium (Friis,
1975).
Experimental design
The experimental design is summarized in Table 1.
Ninety cross-bred piglets free of M. hyopneumoniae and PRRSV were included in the study.
All animals were obtained from a herd that has been free of M. hyopneumoniae and PRRSV
for more than 15 years based on repeated serological testing, and absence of clinical
symptoms and pneumonia lesions. All pigs were selected at one week of age, 40 of them were
randomly vaccinated (v) on the farm with a one-shot commercial vaccine (Stellamune® One,
Pfizer Animal Health). Stellamune is a whole-cell bacterin based on strain P-5722-3 (NL
1042), inactivated with binary ethylamine and oil adjuvanted. Animals were administered 2
ml of the commercial vaccine. The non vaccinated animals were injected with 2 ml of
phosphate buffered saline (PBS), used as a placebo.
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3: EXPERIMENTAL STUDIES
At 21 days of age, piglets were weaned and moved to the experimental facilities of the
Faculty of Veterinary Medicine, Ghent University, Belgium. They were housed in pens with
absolute filters (HEPA U15) and fed ad libitum a commercial feed without antimicrobials.
After one week of acclimatization, the vaccinated piglets were randomly allocated to four
groups. Two groups were intratracheally challenged with the low virulent strain (vLV-4 and
vLV-8) and two with the highly virulent strain (vHV-4 and vHV-8). Forty non-vaccinated
piglets (nv) (n=40) were also assigned to four groups namely two intratracheally challenged
with the low virulent strain (nvLV-4 and nvLV-8) and two with the highly virulent strain
(nvHV-4 and nvHV-8). The remaining ten non-vaccinated piglets were sham-challenged with
sterile culture medium and allocated to groups nvNC-4 and nvNC-8, used as negative
controls.
At 4 weeks post intratracheal inoculation (PI), half of the total number of piglets was
euthanized, whereas the remaining half was euthanized at 8 weeks PI. Deep anaesthesia was
applied prior to euthanasia by administrating intramuscularly 0.3 ml/kg of a mixture of
XylM® 2% and Zoletil 100®, followed by exsanguination. The numbers 4 and 8 in each group
mentioned above designate the weeks (w) PI when piglets were euthanized. The study was
performed after approval of the Ethical Committee for Animal Experiments of the Faculty of
Veterinary Medicine, Ghent University.
Clinical parameters
After intratracheal inoculation, piglets were observed daily for a minimum of 15 minutes. The
severity of coughing was assessed using a respiratory disease score (RDS) as described by
Halbur et al. (1996). Body condition, appetite, presence of dyspnea and tachypnea were also
recorded. The RDS could range from 0 to 6: 0 (no coughing), 1 (mild coughing after an
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3: EXPERIMENTAL STUDIES
encouraged move), 2 (mild coughing in rest), 3 (moderate coughing after encouraged move),
4 (moderate coughing in rest), 5 (severe coughing after encouraged move), 6 (severe coughing
in rest). The daily RDS values were summed and averaged for the two periods namely D28D56 (the first 4 weeks PI) and D56-D84 (the last 4 weeks PI).
Serology
At the age of 7, 28, 42, 56, 70 and 84 days, blood samples were taken from all surviving pigs
and tested for the presence of antibodies against M. hyopneumoniae using the DAKO® Mh
ELISA (Dako Cytomation, Denmark) (Feld et al., 1992). Sera with optical density (OD)values < 50% of the ODbuffer-control were considered positive, OD-values > 50% of the ODbuffercontrol
were classified as negative. On day 28, blood samples were also checked for PRRSV
serum antibodies by means of the HerdChek® PRRS ELISA (Idexx Laboratories, Westbrook,
ME, USA).
Macroscopic lung lesions, histopathology and immunofluorescence (IF) testing for M.
hyopneumoniae
After necropsy, the lungs were removed to score the macroscopic pneumonia lesions
according to Hannan et al. (1982), with values ranging from 0 (no lesions) to 35 (entire lung
affected).
Samples from the right apical, cardial and diaphragmatic lung lobe of each pig were collected
for histopathological examination and IF testing. If lung lesions were present, tissue samples
for IF testing were collected from the edge of a lesion.
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3: EXPERIMENTAL STUDIES
For histopathological examination, the lung tissue samples were fixed in 10% neutral
formalin, processed and embedded in paraffin. After hematoxylin and eosin staining, samples
were scored using light microscopy. Scores could range from 0 to 5, according to the degree
of peribronchiolar and perivascular lymphohistiocytic infiltration, as well as cuffing
formation (Morris et al, 1995; Villarreal et al, 2009). Scores 1 (limited cellular infiltratesmacrophages and lymphocyte- around bronchioles; with airways and alveolar spaces free of
cellular exudates) and 2 (moderate infiltrates with mild diffuse cellular exudates into airways)
were classified as lesions not related to Mycoplasma infections. Scores 3, 4, 5 were
considered to be associated with Mycoplasma infection (mild, moderate or severe- bronchointerstitial (cuffing) pneumonia, surrounding bronchioles but extending to the interstitium,
with lymphofollicular infiltration and mixed inflammatory cell exudates). The average
histopathological lung score per group was calculated.
A direct IF assay (Kobisch et al., 1978) was performed to assess the load of M.
hyopneumoniae organisms in a semi-quantitative way. The scores could range from 0 to 3: 0
(no IF), 1 (limited IF), 2 (moderate IF), and 3 (intense IF) (Vicca et al, 2003).
Nasal swabs, bronchoalveolar lavage fluid, and nPCR
Nasal swabs were collected from all animals just before challenge on day 28 and at euthanasia
(on day 56 and 84).
At necropsy (day 56 or 84), 50 ml of Friis medium was slowly infused in the left lung and
aspirated back into the syringe. The recovered bronchoalveolar lavage (BAL) fluid was
aliquoted, immediately cooled at 4°C, and subsequently stored at -70°C until analysis.
DNA was extracted from the nasal swabs and BAL fluid with the QIAGEN protocol for
purification of total DNA from animal blood and tissues (QIAGEN, DNeasy Blood & Tissue
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3: EXPERIMENTAL STUDIES
Kit, Belgium). For detection of M. hyopneumoniae DNA, a two steps nPCR-test based on a
modified protocol described by Stärk et al. (1998) was performed (Villarreal et al., 2009).
Briefly the nested PCR was performed using Taq polymerase (GIBCO Invitrogen, Belgium)
in a final volume of 20 µl for the first step and 15 µl for the second step. Reaction mixtures
contained PCR buffer (20 mM Tris-HCl (pH 8.4), 50 mM KCl), 3 mM MgCl2, 180 µM of
each dNTP, 0.5 U Taq polymerase and 0.25 μM of each primer MHP950-1L (5'
AggAACACCATCgCgATTTTTA
ATAAAAATggCATTCCTTTTCA
3')
3')
and
(first
step)
MHP950-1R
or
MHP950-2L
(5'
(5'
CCCTTTgTCTTAATTTTTgCAA3') and MHP950-2R (5' gCCgATTCTAgTACCCTAATCC
3') (second step). Two μl of template DNA was used in the first step, 1.5 µl of amplification
product of the first step was used as template in the second step. For both steps, the following
PCR program was used: initial denaturation at 94°C for 2 min; denaturation at 94°C for 30 s,
annealing at 51°C for 30s, and extension at 72°C for 1 min; 35 cycles of denaturation at 94°C
for 20 s, annealing at 55°C for 1 min, and extension at 72°C for 1 min; final extension at 72°C
for 5 min followed by cooling to 10°C. Nested PCR products were analyzed by
electrophoresis on 1.5 % agarose gels in TBE buffer and visualized under UV illumination
after staining in GelRed™ (Biotium. Inc., CA. USA).
Bacteriology
A sample of BAL fluid per pig in each group was inoculated on Columbia agar supplemented
with 5% sheep blood (Oxoid, Hampshire, UK) with a Staphylococcus intermedius streak for
support of Actinobacillus and Haemophilus sp. growth, and on Columbia CNA agar with 5%
sheep blood (Oxoid, Hampshire, UK). As described by Quinn et al. (1994), plates were
incubated overnight in a 5% CO2-enriched environment at 35°C for 48 hours for identification
of respiratory bacteria in the lungs.
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3: EXPERIMENTAL STUDIES
Statistical analysis
One-way analysis of variance (ANOVA) was used to analyse RDS scores, macroscopic and
histopathological lung lesions and IF testing. Levene‘s test was used to assess the
homogeneity of the variances between the different groups. For each parameter, Bonferroni‘s
pair-wise comparisons test was used to evaluate possible differences between groups. This
comparison was made for the two periods 28-56 days (4 weeks PI) and 56-84 days (8 weeks
PI). Statistical results were considered significant when P-values were ≤0.05. The statistical
package SPSS 17.0 for Windows (SPSS 15, SPSS Inc. Illinois, USA) was used to analyse the
data.
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3: EXPERIMENTAL STUDIES
3. Results
Clinical parameters
Three out of the 90 piglets died during the intratracheal inoculation: one from the control
group nvNC-4, one from the nvHV-4 group and one from nvHV-8 group. Necropsies were
performed, but no pathological abnormalities were diagnosed.
The average RDS for the different groups is presented in Table 1. Throughout the entire trial,
coughing was absent in the nvNC-4 and nvNC-8 groups. The onset of coughing in groups
nvLV-4 and nvLV-8 started later (25 DPI) and was milder than in the groups nvHV-4 and
nvHV-8, for which coughing started at ±12 DPI. In the vaccinated groups, the onset of
coughing was observed around the same period as in the corresponding non vaccinated
groups. However, the overall severity of coughing was lower compared to the non vaccinated
groups infected with the same strains.
During the first 4 weeks PI, the highest average RDS value was observed for the nvHV-4
group, which significantly differed from the other groups (P<0.05).
During the 8th week PI, the average RDS values in groups nvLV-8, nvHV-8, vLV-8 and
nvHV-8, were 0.24, 0.44, 0.05 and 0.30, respectively. Significant difference (P<0.05) was
found between the vaccinated groups (vLV-8 and vHV-8) in relation to the respective non
vaccinated groups (nvLV-8 and nvHV-8).
Serology
None of the piglets was serologically positive for M. hyopneumoniae at 7 days of age. The
piglets of the nvNC-4 and nvNC-8 remained negative throughout the study (Table 2).
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3: EXPERIMENTAL STUDIES
In the non vaccinated groups infected with the highly virulent strain, nvHV-4 and nvHV-8,
the first seroconverted pigs were detected on day 42. In the non vaccinated groups infected
with the low virulent strain, nvLV-4 and nvLV-8, seroconverted pigs were detected later (day
56). By the end of the study on day 84, nearly 90% of the animals belonging to the non
vaccinated groups were serologically positive.
In the vaccinated groups, vHV-4, vHV-8 and vLV-4, vLV-8, 100% of the piglets were
serologically positive three weeks after vaccination and remained positive until the day of
euthanasia.
Macroscopic lung lesions, histopathological findings and results of IF testing for M.
hyopneumonia
Macroscopic lung lesions were not observed in the negative control groups, nvNC-4 and
nvNC-8. At 4 weeks PI, significant differences (P<0.05) were found between the different
groups (Table 1). The highest mean values for macroscopic lesions were observed in group
nvHV-4 (6.69), followed by nvLV-4 (2.38); the lowest were in groups vHV-4 (0.11) and
vLV-4 (0.93). At 8 weeks PI, the highest macroscopic lung lesion score was recorded for
group nvHV-8 (5.60), which was significantly different (P<0.05) from the remaining groups:
vHV-8 (1.99), vLV-8 (1.74) and nvLV-8 (1.20).
The mean scores of the histopathological lesions in the different groups at 4 weeks PI were,
from highest to lowest, 4.04 (nvHV-4), 3.10 (vLV-4), 3.02 (vHV-4) and 2.90 (nvLV-4). The
mean score of the nvNC-4 group (1.17) was significantly lower than the mean scores of all
other groups (P<0.05), whereas the mean score of the nvHV-4 group was significantly higher
(P<0.05) than the scores of the other groups (Table 1). At 8 weeks PI, the mean
histopathological scores were 3.62 (nvHV-8), 3.41 (vLV-8), 3.35 (vHV-8) and 2.92 (nvLV142
3: EXPERIMENTAL STUDIES
8). There were no significant differences between groups, apart from the score in the nvNC-8
group, which was the lowest (1.17) (P<0.05).
Apart from the negative control groups, nvNC-4 and nvNC-8, all pigs were positive for IF
staining (Table 2). At 4 weeks PI, there were no statistical differences between vaccinated and
non-vaccinated groups. The highest mean IF score was present in the nvHV-4 (1.26). At 8
weeks PI, the highest IF mean score was recorded in group nvHV-8 (1.44). Group vHV-8
obtained a significantly lower IF score than group nvHV-8 (P< 0.05).
nPCR testing for M. hyopneumoniae on BAL fluid and nasal swabs
The results of the nPCR on BAL fluid and nasal swabs are presented in Table 1. Both non
infected control groups, nvNC-4 and nvNC-8, were negative for nPCR testing on BAL fluid
and nasal swabs. For the other groups, presence of M. hyopneumoniae was confirmed at both
4 and 8 weeks PI.
Bacteriology
At 4 weeks PI, Bordetella bronchiseptica was the only bacterium isolated from BAL fluid in
animals from groups nvLV-4 (3/10), nvHV-4 (1/9), vLV-4 (2/10) and vHV-4 (3/10). At 8
weeks PI, Bordetella bronchiseptica was isolated only in groups nvHV-8 (1/9) and vHV-8
(3/10). Haemophilus parasuis was also isolated from one animal in group nvLV-8.
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3: EXPERIMENTAL STUDIES
Table 1: Clinical, (histo)-pathological observations and presence of M. hyopneumoniae antigens or DNA in the respiratory tract of pigs
intratracheally inoculated with sterile culture medium, a highly or low virulent M. hyopneumoniae strain.
Groups
Nb1
pigs
Vaccinated2
Inoculated3
with
Necropsy at
(weeks PI4)
Average
RDS5
Average lung lesion
score
Macrosc.
nPCR7
Average
IF6 score
Microsc.
BALF8
Nasal swabs
nvLV-4
10
No
LV
4
0.07a
2.38a,b
2.90b
0.77a,b
8
8
nvLV-8
10
No
LV
8
0.24B,D
1.20A
2.92B
1.33B,C
10
10
nvHV-4
10
No
HV
4
0.45b
6.69b
4.04c
1.26b
9
5
nvHV-8
10
No
HV
8
0.44B
5.60B
3.62B
1.44B
8
6
vLV-4
10
Yes
LV
4
0.07a
0.93a
3.10b
0.90a,b
10
8
vLV-8
10
Yes
LV
8
0.05A
1.74A
3.41B
1.33B,C
10
8
vHV-4
10
Yes
HV
4
0.12a
0.11a
3.02b
0.80a,b
10
8
vHV-8
10
Yes
HV
8
0.30C,D
1.99A
3.35B
0.67A,C
10
8
nvNC-4
5
-
medium
4
0.00a
0.00a
1.17a
0.00a
0
0
nvNC-8
5
-
medium
8
0.00A
0.00A
1.17A
0.00A
0
0
Different lowercase letters within a column correspond to significantly different values between the groups at 4w PI; different capital letters
within a column indicate significantly different values between the groups at 8w PI (P< 0.05).
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3: EXPERIMENTAL STUDIES
1
Nb: number
2
Animals were vaccinated at 7 days of age with an inactivated M. hyopneumoniae whole-cell vaccine
3
Animals were intratracheally inoculated at 28 days of age with either sterile culture medium, a highly virulent (HV) or low virulent (LV) M.
hyopneumoniae strain.
4
PI: post-infection
5
RDS: Respiratory disease score
6
IF: Immunofluorescence
7
Number of positive samples by nPCR
8
BALF: bronchoalveolar lavage fluid
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3: EXPERIMENTAL STUDIES
Table 2: Percentage of seropositive pigs (average OD-values) in the different groups at
different time-points during the experimental study. Sera with optical density (OD)-values <
50% of the ODbuffer-control were considered positive, OD-values > 50% of the ODbuffer-control
were classified as negative.
Age of piglets
Group
D7
D28
D42
D56
D70
D84
nvLV-4
0 (93.4)
0 (80.9)
0 (52.0)
50 (43.4)
-
-
nvLV-8
0 (84.4)
0 (85.9)
0 (51.0)
50 (35.7)
80 (56.6)
90 (56.3)
nvHV-4
0 (86.5)
0 (85.9)
11 (21.5)
89 (67.8)
-
-
nvHV-8
0 (84.2)
0 (78.7)
11 (16.1)
78 (47.9)
89 (63.4)
80 (62.4)
vLV-4
0 (82.2)
90 (43.6)
90 (75.1)
90 (93.9)
-
-
vLV-8
0 (73.2)
100 (39.9)
100 (76.9)
100 (101)
100 (104)
100 (107)
vHV-4
0 (82.7)
100 (22.1)
100 (74.1)
100 (108)
-
-
vHV-8
0 (82.7)
100 (20.2)
100 (80.8)
100 (108)
100 (108)
100 (109)
nvNC-4
0 (84.0)
0 (72.3)
0 (13.3)
0 (22.4)
-
-
nvNC-8
0 (88.4)
0 (81.3)
0 (6.9)
0 (16.6)
0 (19.7)
0 (20.1)
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3: EXPERIMENTAL STUDIES
4. Discussion
In corroboration with past findings (Meyns et al., 2007; Vicca et al., 2003), the clinical
symptoms and lung lesions induced by the low virulent M. hyopneumoniae strain were milder
than those caused by the highly virulent strain. Most experimental trials monitor clinical
symptoms and lung lesions only during 4 weeks after challenge. Our results suggest that
although macroscopic lung lesions reached their maximal score within 4 weeks after
challenge, the average RDS in pigs infected with the low virulent strain were higher at 8
weeks than at 4 weeks PI. Thus, a longer period should be considered when studying clinical
symptoms caused by low virulent strains. The understanding of variations in infection
patterns between individual M. hyopneumoniae strains is important for the implementation of
better control measures offering protection against different strains.
The non vaccinated pigs infected with the low virulent strain (nvLV-4 and nvLV-8) had a
slower and lower seroconversion rate than those infected with the highly virulent strain
(nvHV-4 and nvHV-8), very similar to the results obtained by Meyns et al. (2004) in a
transmission study with the same strains. In our trial, two weeks after challenge (42 days of
age), 11% of the pigs belonging to the groups infected with the highly virulent strain had
seroconverted, compared to 0% seroconversion in the groups infected with the low virulent
strain (P< 0.05). The exact reasons for the faster seroconversion in pigs challenged with the
highly virulent strain are not known. It could be due to faster multiplication and induction of a
more severe inflammatory process in the lungs by this strain (Meyns et al., 2007).
All vaccinated pigs (vLV-4, vLV-8, vHV-4 and vHV-8) had seroconverted within three
weeks after vaccination. These results are in agreement with other studies (Sibila et al., 2007),
where a one-shot vaccine during the first week of life induced 100% seroconversion by the
25th day of age.
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3: EXPERIMENTAL STUDIES
In the present study, more positive pigs were detected in BALF samples than in nasal swabs,
confirming that nested PCR on BALF is more suitable as a tool to test infection in individual
animals (Kurth et al., 2002; Sibila et al., 2009).
Vaccination delayed the clinical onset of disease (coughing) and overall significantly reduced
its severity, both in pigs infected with the highly virulent strain and in those infected with the
low virulent strain. The reduction in clinical disease and macroscopic lung lesions was,
however, more prominent in pigs challenged with the highly virulent strain than in pigs
challenged with the low virulent strain. This might be due to the fact that the low virulent
strain only causes mild clinical symptoms and lung lesions are less severe. Hence, the
threshold for improvement by vaccination is lower. At 8 weeks PI, however, significantly
lower IF scores were only demonstrated in vaccinated pigs challenged with the highly virulent
strain and not in the vaccinated animals challenged with the low virulent strain. This might
indicate that the capacity of the vaccine used in the present study to suppress M.
hyopneumoniae multiplication in the lungs is higher after infection with the highly virulent
strain. In the field, variation in efficacy of vaccination strategies between different herds has
also been reported (Maes et al.,1999) and it has been suggested that differences in virulence as
well as divergent protein variability between circulating M. hyopneumoniae strains may be
involved (Calus et al., 2007, Vicca et al., 2003).
In conclusion, the current study demonstrated a difference in infection pattern between the
two M. hyopneumoniae strains both at 4 and 8 weeks PI. Contrary to the highly virulent
strain, the low virulent strain required a period longer than 4 weeks post infection (commonly
accepted as the standard infection model) to induce maximum clinical symptoms. In addition,
vaccination significantly reduced clinical symptoms, macroscopic and microscopic lung
lesions in pigs infected with the highly virulent strain. Protective efficacy was also observed
in pigs infected with the low virulent strain, but the effect was less pronounced.
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3: EXPERIMENTAL STUDIES
Acknowledgements
The study was financially supported by IWT project number 050642. The authors would like
to thank Hanne Vereecke and Alfonso Lopez for all the help and effort dedicated to the
project.
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3: EXPERIMENTAL STUDIES
5. References
Calus, D., Baele, M., Meyns, T., et al. 2007. Protein variability among Mycoplasma
hyopneumoniae isolates. Vet. Microbiol. 120, 284-291.
Ciprian, A., Cruz ,T.A., de la Garza, M., 1994. Mycoplasma hyopneumoniae: interaction with
other agents in pigs, and evaluation of immunogens. Arch Med Res 25, 235-9.
Friis, N., 1975. Some recommendations concerning primary isolation of Mycoplasma
suipneumoniae and Mycoplasma flocculare. A Survey. Nord. Vet.-Med 27, 337-339.
Halbur, P., Paul, P., Meng, X., Lum, M., Andrews, J., Rathje, J., 1996. Comparative
pathogenicity of nine US porcine reproductive and respiratory syndrome virus (PRRSV)
isolates in a 5-week-old cesarean-derived, colostrum-deprived pig model. J. Vet. Diagn.
Invest. 8, 11–20.
Hannan, P.C., Bhogal, B.S., Fish, J.P.., 1982. Tylosin tartrate and tiamutilin effects on
experimental piglet pneumonia induced with pneumonic pig lung homogenate containing
mycoplasmas, bacteria and viruses. Res. Vet. Sci. 33, 76-88.
Kobisch, M., Blanchard, B., Le Potier, M.F., 1993. Mycoplasma hyopneumoniae infection in
pigs: duration of the disease and resistance to reinfection. Vet. Res. 24, 67-77.
Kobisch, M., Pillon, J,. Vannier, P., Magueur, S., and Morvan, P., 1978. Pneumonie
enzootique à Mycoplasma suipneumoniae chez le porc: diagnostic rapide et recherché
d‘anticorps. Recl. Med. Vet. 154, 847–852.
Kurth, K.T., Hsu, T., Snook, E.R., Thacker, E.L., Thacker, B.J., Minion, F.C., 2002. Use of a
Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal
sampling sites in swine. J. Vet. Diagn. Invest. 14, 463-9.
Maes, D., Deluyker, H., Verdonck, M., et al. Effect of vaccination against Mycoplasma
hyopneumoniae in pig herds with an all-in/all-out production system. Vaccine 17, 1024-1034.
Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M., Haesebrouck, F., 2008. Control of
Mycoplasma hyopneumoniae infections in pigs. Vet. Microbiol. 126, 297-309.
150
3: EXPERIMENTAL STUDIES
Meyns, T., Dewulf, J., Calus, D., Haesebrouck, F., Maes, D., 2006. Comparison of
transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations.
Vaccine 24, 7081-7086.
Meyns, T., Maes, D., Calus, D., et al. 2007. Interactions of highly and low virulent
Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs. Vet. Microbiol. 120,
87-95.
Moreau, I.A., Miller, G.Y., Bahnson, P.B., 2004. Effects of Mycoplasma hyopneumoniae
vaccine on pigs naturally infected with M. hyopneumoniae and porcine reproductive and
respiratory syndrome virus. Vaccine 22, 2328-2333.
Morris, C.R., Gardner, I.A.., Hietala, S.K., Carpenter, T.E., Anderson, R.J. and Parker, K.M.,
1995. Seroepidemiologic study of natural transmission of Mycoplasma hyopneumoniae in a
swine herd. Prev. Vet. Med. 21, 323–337.
Morrison, R.B., Pijoan, C., Hilley, H.D., Rapp, V.,1985 Microorganisms associated with
pneumonia in slaughter weight swine. Can. J. Comp. Med. 49, 129-37.
Opriessnig, T., Thacker, E.L., Yu, S., Fenaux, M., Meng, X.J., Halbur, P.G., 2004.
Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual
infection with Mycoplasma hyopneumoniae and porcine circovirus type 2. Vet. Pathol. 41,
624-40.
Pinto, P., Klein, C., Zaha, A., Ferreira, H., 2009. Comparative proteomic analysis of
pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae.
Proteome Science 21, 45.
Quinn, P., Carter, M., Markey, B., Carter, G. (Eds.) Clinical Vet. Microbiol.. Edinburgh,
Mosby, 1994: 254–259.
Ross, R.F. Mycoplasmal diseases. In: Leman AD, Straw BE, Mengeling WL, Allaire SD,
Taylor DJ, editors. Diseases of Swine, eighth ed. Iowa, Iowa State University Press, 1999:
537–551.
Sibila, M., Nofrarias, M., Lopez-Soria, S., et al. 2007. Exploratory field study on Mycoplasma
hyopneumoniae infection in suckling pigs. Vet. Microbiol. 121, 352-356.
151
3: EXPERIMENTAL STUDIES
Sibila M, Pieters M, Molitor T, Maes D, Haesebrouck F, Segales J. 2009. Current
perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection.
Vet. J. 181, 221-31.
Stakenborg, T., Vicca, J., Butaye, P., et al. 2005. The diversity of Mycoplasma
hyopneumoniae within and between herds using pulsed-field gel electrophoresis. Vet.
Microbiol. 109, 29-36.
Stakenborg, T., Vicca, J., Maes, D., et al. 2006. Comparison of molecular techniques for the
typing of Mycoplasma hyopneumoniae isolates. J. Microbiol. Methods 66, 263-75.
Thacker, E.L., Halbur, P.G., Ross, R.F., Thanawongnuwech. R., Thacker, B.J., 1999.
Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome
virus-induced pneumonia. J. Clin. Microbiol. 37, 620-627.
Thacker, E.L., Thacker, B.J., Boettcher, T.B., et al. 1998. Comparison of antibody production,
lymphocyte stimulation, and protection induced by four commercial Mycoplasma
hyopneumoniae bacterins. J. Swine Health Prod. 6, 107–112.
Vicca, J., Stakenborg, T., Maes, D., et al. 2003. Evaluation of virulence of Mycoplasma
hyopneumoniae field isolates. Vet. Microbiol. 30, 177-190.
Villarreal, I., Maes, D., Meyns, T., Gebruers, F., Calus, D., Pasmans F., Haesebrouck, F.,
2009. Infection with a low virulent Mycoplasma hyopneumoniae isolate does not protect
piglets against subsequent infection with a highly virulent M. hyopneumoniae isolate. Vaccine
27, 1875-9.
Whittlestone, P. 1972. The role of mycoplasmas in the production of pneumonia in the pig.
In: Pathogenic Mycoplasmas. Amsterdam, Associated Scientific Publishers, p. 263-283.
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3.4 Infection with a low virulent Mycoplasma hyopneumoniae isolate does
not protect piglets against subsequent infection with a highly virulent M.
hyopneumoniae isolate
I.Villarreala, D. Maesa, T. Meynsb, F. Gebruersb, D. Calusb, F. Pasmansb, and F. Haesebrouckb
a
Department of Reproduction, Obstetrics and Herd Health. Veterinary Epidemiology Unit
Ghent University, Faculty of Veterinary Medicine. Salisburylaan 133, B-9820 Merelbeke,
Belgium.
b
Department of Pathology, Bacteriology and Poultry Diseases. Ghent University, Faculty of
Veterinary Medicine. Salisburylaan 133, B-9820 Merelbeke, Belgium.
Vaccine, 27, 2009 (12): 1875-1879
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Abstract
The study aimed to evaluate the effect of an infection with low virulent isolates of M.
hyopneumoniae (LV1 and LV2) on the subsequent infection with a highly virulent isolate
(HV). Fifty-five, 3 week-old piglets free of M. hyopneumoniae were randomly allocated to 6
different groups. At 4 weeks of age (D0), groups LV1-HV and LV1 were intratracheally
inoculated with LV1, groups LV2-HV and LV2 with LV2, and group HV with sterile culture
medium. Four weeks later (D28), the pigs of these different groups were either intratracheally
inoculated with the highly virulent isolate (groups LV1-HV, LV2-HV, HV) or with sterile
culture medium (groups LV1 and LV2). A negative control group consisted of pigs inoculated
with sterile culture medium on D0 and D28. All animals were necropsied at 28 days after the
second inoculation (D56). Clinical symptoms were evaluated daily using a respiratory disease
score (RDS). After necropsy, macroscopic and histopathological lung lesions were quantified
and immunofluorescence (IF) testing on lung tissue and nested PCR on BAL fluid were
performed for the detection of M. hyopneumoniae.
Disease signs and lung lesions were not observed in pigs of the negative control group. In the
other groups, there were no or only very mild clinical symptoms from D0 until D28. A
significant increase in the average RDS values was, however, observed during D28-D56,
especially in groups LV1-HV (1.48) and LV2-HV (1.49), in group HV (0.79), and to a lesser
extent in groups LV1 (0.50) and LV2 (0.65) (P<0.05). The clinical symptoms during D28D56, the lung lesions and intensity of IF staining were more pronounced in groups LV1-HV,
LV2-HV and HV compared to groups LV1 and LV2. All pigs, except those from the negative
control group, were positive on IF testing and PCR at D56. The present study demonstrates
that pigs inoculated with low virulent isolates of M. hyopneumoniae are not protected against
a subsequent infection with a highly virulent isolate 4 weeks later and may even develop more
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severe disease signs. This indicates that subsequent infections with different M.
hyopneumoniae isolates may lead to more severe clinical disease in a pig herd.
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1. Introduction
Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary cause of enzootic
pneumonia in pigs, a chronic respiratory disease that causes major losses to the pig industry
worldwide (Thacker et al., 1999). The infection pattern and the severity of the disease are
largely determined by management practices and housing conditions (Maes et al., 2008), but
also the virulence of the M. hyopneumoniae isolate is important (Vicca et al., 2003; Meyns et
al., 2004). Experimental infections with a highly virulent isolate consistently resulted in
clinical disease and lung lesions, whereas infection with a low virulent isolate was associated
with no or very mild clinical symptoms and lung lesions (Vicca et al., 2003; Meyns et al.,
2006).
Previous studies showed that M. hyopneumoniae isolates originating from the same batch of
slaughter pigs were very similar at genomic level, whereas isolates originating from different
batches showed major diversity at genomic (Stakenborg et al., 2005) and also at proteomic
level (Calus et al., 2007). Since infected pigs are commonly transferred from one herd to
another, it can be expected that different M. hyopneumoniae isolates are circulating within the
same herd. This would imply that under field conditions, pigs may become infected with
different M. hyopneumoniae isolates before they reach slaughter age. The effect of multiple
infections with different M. hyopneumoniae isolates on the severity of the disease is, however,
not known.
The currently available inactivated whole-cell vaccines do not offer complete protection
against lung lesions, they do not prevent colonization (Thacker et al., 1999) and they are not
able to significantly reduce the transmission of M. hyopneumoniae (Meyns et al., 2006).
Consequently, they are not suitable to eliminate M. hyopneumoniae from infected pig herds
neither to end up in a sustainable control of the disease. Attenuated vaccines might be a good
alternative. Indeed, for other Mycoplasma sp. such as M. synoviae in chickens, live
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attenuated, temperature sensitive vaccines have been used successfully to control virulent
infection (synovitis and respiratory disease) in commercial chicken flocks (Jones et al., 2006).
For M. hyopneumoniae it is not known if infection with low virulent isolates may induce
protection against highly virulent isolates.
The present study aimed to investigate the effect of infection of pigs with low virulent isolates
of M. hyopneumoniae on a subsequent challenge infection with a highly virulent isolate.
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2. Materials and methods
Animals
Fifty-five, 3 week-old, cross-bred (Rattlerow Seghers®, Buggenhout, Belgium) piglets were
weaned and moved to the animal facilities of the Faculty of Veterinary Medicine, Ghent
University, Belgium. The piglets were obtained from a herd that was free of M.
hyopneumoniae and PRRSV. The herd had been monitored repeatedly during the last 8 years
by means of clinical, pathological and serological parameters for M. hyopneumoniae and
PRRSV in sows and pigs of different age categories, and none of the results showed evidence
for presence of these pathogens. The piglets were acclimatized during one week, and during
the entire trial, they were fed a commercial feed with no antimicrobials.
Experimental design and M. hyopneumoniae isolates
After acclimatization, when the pigs were 28 days old (D0), they were randomly allocated to
6 different groups (Table 1). Each group was housed in a different compartment that was
equipped with absolute filters (HEPA U15) to avoid spread of the pathogen between groups.
Two different low virulent isolates namely F1 (LV1) and F13 (LV2), and one highly virulent
isolate namely F7 (HV) were used for the inoculations. The virulence of these isolates had
been tested in previous experimental infection models (Vicca et al., 2003). At D0, groups
LV1-HV and LV1 were inoculated with LV1, groups LV2-HV and LV2 with LV2, and group
HV with sterile culture medium. Four weeks later (D28), the pigs of these different groups
were either intratracheally inoculated with the highly virulent isolate (groups LV1-HV, LV2HV, HV) or with sterile culture medium (groups LV1 and LV2). A negative control group
consisted of pigs inoculated with sterile culture medium on D0 and D28. For the inoculations,
the pigs were anaesthetized with 0.22 ml/kg of a mixture of Xyl-M® 2% (Intervet) and Zoletil
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100® (Virbac). Depending on the group, they were inoculated intratracheally with either 7 ml
of 107 color changing units (CCU) per ml of a low or highly virulent M. hyopneumoniae
isolate, or with 7 ml of sterile culture medium.
All animals were euthanized and necropsied at 28 days after the second inoculation (D56).
Therefore, deep anaesthesia was applied by administrating intramuscularly 0.3 ml/kg of a
mixture of XylM® 2% and Zoletil 100®, followed by exsanguination.
Table 1: The different groups used to study the effect of infection with low virulent M.
hyopneumoniae isolates (LV1 and LV2) on subsequent challenge infection with a highly
virulent (HV) isolate. The first inoculation took place when the pigs were 4 weeks old (D0)
and the second inoculation 4 weeks later (D28). All pigs were euthanized at 4 weeks after the
second inoculation (D56).
Group
Number of
animals
Inoculation at D0 with
Inoculation at D28
with
LV1
10
LV1
sterile medium
LV2
10
LV2
sterile medium
LV1-HV
10
LV1
HV
LV2-HV
10
LV2
HV
HV
10
sterile medium
HV
control
5
sterile medium
sterile medium
Clinical parameters
The piglets were observed daily for a minimum of 15 minutes throughout the entire study
period (D0-D56). The severity of coughing was graded in a blinded manner by means of a
respiratory disease score (RDS) (Halbur et al., 1996). The RDS could range from 0 to 6: 0 (no
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3: EXPERIMENTAL STUDIES
coughing), 1 (mild coughing after an encouraged move), 2 (mild coughing while being in
rest), 3 (moderate coughing after encouraged move), 4 (moderate coughing while being in
rest), 5 (severe coughing after encouraged move), 6 (severe coughing while being in rest). The
daily RDS values were summed and averaged for the two periods namely D0-D28 and D28D56.
Macroscopic and histopathological lung lesions, and immunofluorescence (IF) testing for M.
hyopneumoniae
After necropsy, the lungs were removed and a digital picture was taken from all the lungs for
further reference. The macroscopic pneumonia lesions were quantified in a blinded manner
using the lung lesion score as described by Hannan et al. (1982). Values could range
theoretically from 0 (no lesions) to 35 (entire lung affected).
Lung tissue samples for histopathological examination and IF testing were taken from each
apical, cardial and diaphragmatic lobe from each pig. In case lung lesions were present, tissue
samples for IF testing were collected from the edge of a lesion.
The tissue samples for histopathological examination were fixed in 10% neutral formalin,
processed and embedded in paraffin. One slide was made per lobe sample. Peribronchiolar
and perivascular lymphohistiocytic infiltration and nodule formation consistent with M.
hyopneumoniae lesions were scored (0-5) using light microscopy as described previously
(Vicca et al., 2003). Scores 1 (limited cellular infiltrates- macrophages and lymphocytearound bronchioles; with airways and alveolar spaces free of cellular exudates), and 2
(moderate infiltrates with mild diffuse cellular exudates into airways) were classified as
lesions not related to Mycoplasma infections. Scores 3, 4, 5 were considered to be associated
with Mycoplasma infection (mild, moderate or severe- broncho-interstitial (cuffing)
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3: EXPERIMENTAL STUDIES
pneumonia, surrounding bronchioles but extending to the interstitium, with lymphofollicular
infiltration and mixed inflammatory cell exudates) (Morris et al, 1995). One measurement per
lobe and three lobes per pig were investigated. The average score per inoculation group was
calculated.
IF testing for M. hyopneumoniae was done using the assay developed by Kobisch (Kobisch et
al., 1978), and the result was expressed semi-quantitatively using scores ranging from 0 to 3:
0 (no IF), 1 (limited IF), 2 (moderate IF), and 3 (intense IF) (Vicca et al., 2003)
Serology
Blood samples were taken from all pigs at D0, D14, D28, D42 and D56 and tested for the
presence of antibodies against M. hyopneumoniae using the DAKO® Mh ELISA (Dako
Cytomation, Denmark) (Feld et al., 1992). Sera with optical density (OD)-values < 50% of the
ODbuffer-control were considered positive, OD-values > 50% of the ODbuffer-control were classified
as negative. Intermediate OD-values were considered doubtful and classified as negative.
BAL fluid and nPCR
The lungs were removed during necropsy, and bronchoalveolar lavage (BAL) fluid was
collected from the left part of the lungs before taking the samples for histopathology and IF.
A total of 50 ml of Friis fluid was slowly infused in the lung and aspirated back into the
syringe. The recovered fluid was aliquoted, immediately cooled at 4°C, and subsequently
stored at -70°C until analysis.
DNA was extracted from BAL fluid with the QIAGEN protocol for purification of total DNA
from animal blood and tissues (QIAGEN, DNeasy Blood & Tissue Kit, Belgium). For
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detection of M. hyopneumoniae DNA, a nPCR-test based on the R1 repeat of P97 was used
enabling discrimination between the different M. hyopneumoniae isolates used for infection
(Gebruers et al., 2008).
Statistical analysis
The RDS scores, macroscopic and histopathological lung lesions, percentage of air in the
lungs, IF testing and serology were evaluated by using one-way analysis of variance
(ANOVA). Levene‘s test was used to assess the homogeneity of the variances between the
different groups. For each parameter, pair-wise comparisons between groups were made using
Bonferoni‘s test. RDS scores during D0-D28 and D28-D56 were compared within each group
using paired t-tests. Statistical results were considered significant when P-values were ≤0.05.
The statistical package SPSS 15.0 for Windows (SPSS 15, SPSS Inc. Illinois, USA) was used
to analyse the data.
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3. Results
Clinical parameters
Three out of the 55 piglets died during the study: one from the LV1-HV group (at D14), one
from the LV2 group (at D18) and one from the control group (at D18). Necropsies were
performed to establish the causes of death. The piglet from the LV1-HV group died due to a
congenital stenosis of the rectum, the two other piglets died due to meningitis caused by
Streptococcus suis serotype 2 infection.
During D0-D28, coughing was absent in the groups that were inoculated with sterile culture
medium (group HV, negative control group), and was very mild in the 4 other groups that
were infected with a low virulent isolate (Figure 1). Very mild coughing started first in the
LV2 group between D7-D10.
During D28-D56, coughing was present in all groups, except for the control group. The RDS
values were significantly higher during D28-D56 compared to D0-28 in all infected groups
(P<0.05). The highest average RDS values were observed in groups LV1-HV (1.48) and LV2HV (1.49), the lowest in groups LV1 (0.50) and LV2 (0.64). Intermediate RDS values (0.79)
were observed in group HV (Figure 1). The average RDS value was significantly lower in the
LV1 group compared to the other infected groups (P<0.05). During the period D28-D56, the
severity of coughing increased towards the end of the study.
Macroscopic and histopathological lung lesions, and IF testing for M. hyopneumoniae
Macroscopic lung lesions were not observed in the negative control group. Only numerical
differences (P>0.05) were found between the different infected groups (Table 2). The highest
values for macroscopic lesions were observed in group LV1-HV (4.14), HV (3.49) and LV2HV (2.53), the lowest in groups LV1 (1.92) and LV2 (2.17).
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The mean scores of the histopathological lesions in the different groups were 2.27 (LV1),
2.67 (LV2), 3.44 (LV1-HV), 3.17 (LV2-HV), and 3.10 (HV). The mean scores of LV1-HV
and LV2-HV were significantly different from those of the LV1 group (P<0.05). The mean
score of the control group (0.42) was significantly lower than the mean scores of all other
groups (P<0.05) (Table 2). In pigs of the LV1 and LV2 groups, lymphohistiocytic infiltration
was predominantly seen in the pulmonary interstitial space, whereas in pigs of the HV group
it mainly surrounded the airways. The infiltration in pigs of groups LV1-HV and LV2-HV
was present in the pulmonary interstitial space as well as around the airways.
All pigs were positive for IF staining, except for those of the negative control group. The
highest mean IF scores were present in the LV1-HV (1.92) and LV2-HV (1.97) groups, but
there were no significant differences with the mean scores of the other infected groups (LV1
1.53; LV2 1.48; HV 1.57).
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3: EXPERIMENTAL STUDIES
Table 2: Results (average  SD) of macroscopic and histopathological (lymphohistiocytic
infiltration) lung lesion scores and immunofluorescence (IF) scores in the different groups
(LV low virulent; HV highly virulent)
1
Group
Macroscopic lung
lesions1
Histopathological lung
lesions1
IF1
LV1
1.92a ± 1.6
2.27b ± 0.5
1.53a ± 0.7
LV2
2.17a ± 1.8
2.67ab ± 0.7
1.48a ± 0.6
LV1+HV
4.14a ± 2.3
3.44a ± 0.7
1.92a ± 0.4
LV2+HV
2.53a ± 2.1
3.17a ± 0.5
1.97a ± 0.3
HV
3.49a ± 3.3
3.10ab ± 0.7
1.57a ± 0.5
control
0.00b ± 0.0
0.42c ± 0.2
0.00b ± 0.0
Values with different superscripts within a column are significantly different (P<0.05)
Serology
In none of the piglets antibodies to M. hyopneumoniae were detected at D0, and the pigs of
the negative control group remained negative throughout the entire study (Table 3). The
percentage of serologically positive pigs 4 weeks after infection (D28) was 0% for the LV1
and LV1-HV groups, and 33% and 30% for the LV2 and LV2-HV groups, respectively. The
percentage of serologically positive pigs at D56, namely 8 weeks after infection with a low
virulent isolate, increased to 30% (LV1) and 89% (LV2). Ninety (90) percent of the pigs of
the HV group were serologically positive 4 weeks after infection (D56). In almost all pigs in
the groups with the combined infections, antibodies to M. hyopneumoniae were detected at
D56.
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Table 3: Percentage of seropositive pigs (average OD-values) in the different groups at
different time-points during the experimental study (D0-D56) (LV low virulent; HV highly
virulent)
D0
D14
D28
D42
D561
LV1
0 (101)
0 (85.7)
0 (74.9)
10 (78.3)
30 (64.3)a
LV2
0 (89.1)
0 (70)
33 (64.9)
78 (34.8)
89 (19.6)b
LV1-HV
0 (92.2)
0 (85.6)
0 (70.9)
67 (36.1)
89 (18.1)b
LV2-HV
0 (94.4)
14 (71.4)
30 (55.1)
80 (31.9)
100 (14.4)b
HV
0 (94.4)
0 (82.9)
0 (82.1)
10 (69.8)
90 (35)b
control
0 (96.2)
0 (85.3)
0 (97.7)
0 (86)
0 (92.7)c
1
Values with different superscripts at D56 are significantly different (P<0.05)
nPCR testing on BAL fluid
All piglets from the negative control group were negative using nPCR testing on BAL fluid,
all other pigs were positive. In the LV1-HV group, the low virulent and highly virulent isolate
were detected in 7/9 and 9/9 of the pigs, respectively. In the LV2-HV group, the low and
highly virulent isolate was detected in 7/10 and 10/10 of the pigs, respectively.
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4. Discussion
The present study showed that infection with a low virulent M. hyopneumoniae isolate does
not protect piglets against subsequent infection 4 weeks later with a highly virulent isolate. It
appeared, based on clinical parameters and macroscopic and histopathological lesions, that a
previous infection with a low virulent isolate may increase the severity of an infection with a
highly virulent isolate.
The two low virulent isolates did only induce very mild clinical symptoms during the first 4
weeks after experimental infection. This confirms the results of previous experimental studies
with these isolates (Vicca et al., 2003). However, from 4 until 8 weeks post infection, the pigs
of the LV1 and LV2 groups developed more severe clinical symptoms and showed mild to
moderate macroscopic and microscopic lung lesions. Consequently, it appears that disease
signs develop later after infections with these isolates compared to highly virulent isolates.
The findings also suggest that the widely used experimental infection model for M.
hyopneumoniae namely monitoring of the clinical symptoms during 4 weeks combined with
evaluating the lung lesions at 4 weeks after challenge infection is not well suited to assess the
full virulence capacity of such isolates. The standard infection model for M. hyopneumoniae
is based on the observation that lung lesions reach their maximal size 2 to 4 weeks after
experimental infection (Zielinski et al., 1992; Kobisch et al., 1993). The effect of the
inoculation with sterile isolation medium at 28 days after infection with the low virulent
isolate is, however, not known and it can not be excluded that this may have influenced the
disease outcome.
The percentage of seropositive pigs infected with the low virulent isolates was significantly
higher (P<0.05) at D56 (8 weeks after infection) compared to D28 (4 weeks after infection),
but the response in group LV1 was significantly lower than in pigs subsequently infected with
the highly virulent isolate. In a study designed to quantify the transmission of M.
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hyopneumoniae, Meyns et al. (2006) also found that pigs infected with a low virulent isolate
had a lower and slower serological response compared to pigs infected with a highly virulent
isolate. The precise reason for this is not known, but it is possible that low virulent M.
hyopneumoniae isolates multiply slower in the lung tissue after infection, and that as a result
of this, they trigger the immune response less effectively. Pigs of the LV1 and LV2 groups
had the lowest IF scores, suggesting that indeed less M. hyopneumoniae organisms were
present in the lung tissue. Young et al. (2000) also stated that highly virulent isolates had a
higher capacity to colonize or attach to the ciliated epithelium.
The results confirm that detecting serum antibodies for M. hyopneumoniae is not a reliable
tool to assess the infection status of an individual animal (Fano et al., 2005). Even the present
experimental procedure in which pigs were inoculated intratracheally with 7 x 107CCU of M.
hyopneumoniae did not elicit detectable serum antibodies 8 weeks later in 40% of the
animals. It also appeared that the serological response was quite variable between the LV1
and LV2 group. At D56, serum antibodies were detected in 30% and 89% of the pigs
inoculated with LV1 and LV2, respectively.
The results of the HV group were very similar to those observed in previous experiments with
this isolate (Vicca et al., 2003; 2005), confirming the virulence of the isolate and the
reproducibility of the experimental infection model used. There were no indications of a
protective immunity induced by infection with the low virulent isolates on subsequent
infection with the highly virulent isolate. RDS from D28 to D56, microscopic lung lesions,
and the intensity of IF staining in the lungs were significantly or numerically higher for the
groups with a combined infection (LV1-HV; LV2-HV) than for groups inoculated with either
only a low or highly virulent isolate. This suggests that low virulent isolates, although they do
not induce severe clinical symptoms by themselves, may increase the severity of subsequent
infection with a highly virulent isolate. The exact mechanisms for this interaction are not
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known. It may be related to a more pronounced inflammatory reaction of the host, as the
inflammatory response is known to be involved in the clinical symptoms and lung lesions
(Thacker et al., 2006).
In the current experiment, pigs were intratracheally inoculated with high doses of M.
hyopneumoniae. Under field conditions, infection doses are not known but they are probably
lower than the 7 x 107 CCU used in this study. The higher inoculation dose in our experiment
may result in a faster and a higher level of colonization at the surface of the airways,
compared to field infections. This agrees with the observation of a faster induction of
seroconversion in experimentally inoculated animals compared to contact infected animals
(Feld et al., 1992; Fano et al., 2005). On the other hand, the housing conditions, management
practices and air quality in the experimental setting were better than those present under field
conditions. Also, infections with pathogens like PRRSV were not present in our experimental
animals. These infections may influence the clinical outcome of M. hyopneumoniae infections
(Thacker et al., 1999). Although extrapolation of results obtained in experimentally infected
pigs to the field situation should be done with caution, the infection model used allows
studying the effects of infections with M. hyopneumoniae of different virulence in a
standardized and reproducible way.
In conclusion, the present study demonstrates that pigs inoculated with low virulent isolates of
M. hyopneumoniae isolates are not protected against a subsequent infection with a highly
virulent isolate 4 weeks later and may even develop more severe disease signs. This indicates
that subsequent infections with different M. hyopneumoniae isolates may lead to more severe
clinical disease in a pig herd. Further research is necessary to assess the precise mechanisms
influencing the interactions between highly and low virulent isolates, and to investigate the
importance of the present findings under field conditions.
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Acknowledgements
The study was financially supported by IWT project number 050642. The authors would like
to thank Hanne Vereecke for all the help, effort and laboratory work dedicated to the project.
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5. References
Akira, S., Hemmi, H., 2003. Recognition of pathogen-associated molecular patterns by TLR
family. Immunol. Lett. 85, 85–95.
Artiushin, S., Minion, F.C., 1996. Arbitrarily primed PCR analysis of Mycoplasma
hyopneumoniae field isolates demonstrates genetic heterogeneity, Int. J. Syst. Bacteriol. 46,
324–328.
Asai, M., Okada, M., Ono, T., Irisawa, Y., Mori, Y., Yokomizo, Sato, S., 1993. Increased
levels of tumor necrosis factor and interleukin 1 in bronchoalveolar lavage fluids from pigs
infected with Mycoplasma hyopneumoniae, Vet. Immunol. Immunopathol. 38, 253–260.
Asai, T., Okada, M., Ono, M., Mori, Y., Yokomizo, Sato, S., 1994. Detection of interleukin-6
and prostaglandin E2 in bronchoalveolar lavage fluids of pigs experimentally infected with
Mycoplasma hyopneumoniae, Vet. Immunol. Immunopathol. 44, 97–102.
Baranyi, J., and Roberts, T.A., 1996. A dynamic approach to predicting bacterial growth in
food, Int. J. Food. Microbiol. 23, 277–293.
Blanchard, B., Vena, M.M., Cavalier, A., Le Lannic, J., Gouranton, J. Kobisch, M., 1992.
Electron microscopic observation of the respiratory tract of SPF piglets inoculated with
Mycoplasma hyopneumoniae, Vet. Microbiol. 30, 329–341.
Calsamiglia, M., Pijoan, C., Bosch, G., 1999. Profiling Mycoplasma hyopneumoniae in farms
using serology and a nested PCR technique, Swine Health and Prod. 7, 263-268.
Calus, D., Baele, M., Meyns T, de Kruif A, Butaye P, Decostere A, Haesebrouck F, Maes D.,
2007. Protein variability among Mycoplasma hyopneumoniae isolates. Vet. Microbiol. 120,
284-91.
Choi, C., Kwon, D., Jung, K., Ha, Y., Lee, Y.H., Kim, O., Park, H.K., Kim, S.H., Hwang,
K.K. and Chae, C., 2006. Expression of inflammatory cytokines in pigs experimentally
infected with Mycoplasma hyopneumoniae, J. Comp. Path. 134, 40–46.
Cole, B.C., 1991. The immunobiology of Mycoplasma arthritidis and its superantigen MAM,
Curr. Top. Microbiol. 174, 107–119.
171
3: EXPERIMENTAL STUDIES
Deiters, U., and Mülhradt, P.F., 1999. Mycoplasmal lipopeptide MALP-2 induced the
chemoattractant
proteins
macrophage
inflammatory protein
1
(MIP-1),
monocyte
chemoattractant protein 1, and MIP-2 and promotes leukocyte infiltration in mice, Infect.
Immun. 67, 3390–3398.
Fano, E., Pijoan, C., Dee, S., 2005. Dynamics and persistance of Mycoplasma hyopneumoniae
infection in pigs. Can. J. Vet. Res. 69, 223-228.
Feld, N., Qvist, P., Ahrens, P., Friis, N., Meyling, A., 1992. A monoclonal blocking ELISA
detecting serum antibodies to Mycoplasma hyopneumoniae, Vet. Microbiol. 30, 35–46.
Frey, J., Haldimann, A., Nicolet, J., 1992. Chromosomal heterogeneity of various
Mycoplasma hyopneumoniae field isolates, Int. J. Syst. Bacteriol. 42, 275–280.
Friis, N.F. 1975. Some recommendations concerning primary isolation of Mycoplasma
suipneumoniae and Mycoplasma flocculare a survey, Nord. Vet. Med. 27, 337–339.
Gebruers, F., Calus, D., Maes, D., Villarreal, I., Pasmans, F., Haesebrouck, F. A set of four
nested pcrs to detect different strains of Mycoplasma hyopneumonaie in biological samples.
17th IOM Congress. 2008.
Halbur, P.C., Paul, P.S., Meng, X.J., Lum, M.A., Andrews, J.J., Rathje, J.A., 1996.
Comparative pathogenicity of nine US Porcine Reproductive and Respiratory Syndrome
Virus (PRSSV) isolates in a five-week-old cesarean-derived, colostrumdeprived pig model. J
Vet. Diagn. Inv. 8, 11-20.
Hannan, P.C., Bhogal, B.S., Fish, J.P., 1982. Tylosin tartrate and tiamutilin effects on
experimental piglet pneumonia induced with pneumonic pig lung homogenate containing
mycoplasmas, bacteria and viruses, Res. Vet. Sci. 33, 76–88.
Hsu, T., Minion, F.C., 1998. Identification of the cilium binding epitope of the Mycoplasma
hyopneumoniae P97 adhesin, Infect. Immun. 66, 4762–4766.
Into, T., Kiura, K., Yasuda, M., Kataoka, H., Inoue, N., Hasebe, A., Takeda, K., Akira, S.,
Shibata, K., 2004. Stimulation of human Toll-like receptor (TLR)2 and TLR6 with membrane
lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-κB activation,
Cell. Microbiol. 6, 187–199.
172
3: EXPERIMENTAL STUDIES
Jones, J.F., Whithear, K.G., Scott, P.C., Noormohammadi, A.H., 2006. Duration of immunity
with Mycoplasma synoviae: comparison of the live attenuated vaccine MS-H (Vaxsafe MS)
with its wild-type parent strain, 86079/7NS. Avian Dis. 50, 228-31
Kobisch, M., Pillon, J., Vannier, P., Magueur, S., Morvan, P., 1978. Pneumonie enzootique à
Mycoplasma suipneumoniae chez le porc: diagnostic rapide et recherché d‘anticorps, Recl.
Med. Vet. 154, 847–852.
Kobisch, M., Blanchard, B., Portier, M., Le Potier, M., 1993. Mycoplasma hyopneumoniae
infection in pigs: duration of the disease and resistance to reinfection. Vet. Res. 24, 67-77.
Lührmann, A., Deiters, U., Skokowa, J., Hanke, M., Gessner, J.E., Mülhradt, P.F., Pabst, R.,
Tschernig, T., 2002. In vivo effects of a synthetic 2-kilodalton macrophage activating
lipopeptide of Mycoplasma fermentans after pulmonary application, Infect. Immun. 70, 3785–
3792.
Maes D, Verdonck M, Deluyker H and De Kruif A., 1996. Enzootic pneumonia in pigs, Vet.
Quart.18, 104–109.
Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M., Haesebrouck, F., 2008. Control of
Mycoplasma hyopneumoniae infections in pigs. Vet. Microbiol.. 126, 297-309
Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F., De Kruif, A., 2004.
Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using
transmission experiments, Prev. Vet. Med. 66, 265–275.
Meyns, T., Dewulf, J., de Kruif, A., Calus, D., Haesebrouck, F., Maes, D., 2006. Comparison
of transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated
populations. Vaccine 24, 7081-6
Meyns, T., Maes, D., Calus, D., Ribbens, S., Dewulf, J., Chiers, K., de Kruif, A., Cox, E.,
Decostere, A., Haesebrouck, F., 2007. Interactions of highly and low virulent Mycoplasma
hyopneumoniae isolates with the respiratory tract of pigs. Vet. Microbiol.. 120, 87-95
Minion, F.C., Adams, C., Hsu, T., 2000. R1 region of P97 mediates adherence of
Mycoplasma hyopneumoniae to swine cilia. Infect. Immun. 68, 3056-60
173
3: EXPERIMENTAL STUDIES
Minion, F.C., Lefkowitz, E.J., Madsen, M.L., Cleary, B.J., Swartzell, S.M., Mahairas, G.G.,
2004. The genome sequence of Mycoplasma hyopneumoniae isolate 232, the agent of swine
mycoplasmosis, J. Bacteriol. 186, 7123–7133.
Morris, C.R., Gardner, I.A., Hietala, S.K., Carpenter, T.E., Anderson, R.J., Parker, K.M.,
1995. Seroepidemiologic study of natural transmission of Mycoplasma hyopneumoniae in a
swine herd, Prev. Vet. Med. 21, 323–337.
Mühlradt PF, Kieβ M, Meyer H, Süßmuth R and Jung G, 1997. Isolation, structure
elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma
fermentans acting at picomolar concentration. J. Exp. Med. 185, 1951–1958.
Mühlradt, P.F., Kie, M., Meyer, H., Süßmuth, R., Jung, G., 1998. Structure and specific
activity of macrophage-stimulating lipopeptides from Mycoplasma hyorhinis. Infect. Immun.
66, 4804–4810.
Okusawa, T., Fujita, M., Nakamura, J., Into, T., Yasuda, M., Yoshimura, A., Hara, Y.,
Hasebe, A., Golenbock, D.T., Morita, M., Kuroki, Y., Ogawa, T., Shibata, K., 2004.
Relationship between structures and biological activities of mycoplasmal diacylated
lipopeptides and their recognition by Toll-like receptors 2 and 6, Infect. Immun.72, 1657–
1665.
Ro, L. and Ross, R.F., 1983. Comparison of Mycoplasma hyopneumoniae isolates by
serologic methods, Am. J. Vet. Res. 44, 2087–2094.
Rodríguez, F., Ramírez, G.A., Sarradell, J., Andrada, M. and Lorenzo, H., 2004.
Immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with
Mycoplasma hyopneumoniae, J. Comp. Path. 130, 306–312.
Smart, S.J. and Casale, T.B., 1994. Pulmonary epithelial cells facilitate TNF-alpha-induced
neutrophil chemotaxis. A role for cytokine networking, J. Immunol. 152, 4087–4094.
Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Peeters, J., De Kruif, A., Haesebrouck, F.,
2005. Diversity of Mycoplasma hyopneumoniae within and between herds using pulsed-field
gel electrophoresis, Vet. Microbiol. 109, 29–36.
174
3: EXPERIMENTAL STUDIES
Stakenborg, T., Vicca, J., Maes D, Peeters J, de Kruif A, Haesebrouck F and Butaye P., 2006.
Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates,
J. Microbiol. Meth. 66, 263–275.
Thacker, E.L., Halbur, P.G, Ross, R.F., Thanawongnuwech, R., Thacker, B.J., 1999.
Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome
virus-induced pneumonia. J Clin Micorbiol. 37, 620–627.
Thacker E. Mycoplasmal diseases. In: A.D. Leman, B.E. Straw, S. D‘Allaire, W.L.
Mengeling and D.J. Taylor, Editors, Diseases of Swine, The Iowa State University Press,
Ames, IA. 2006: 701–717.
Van Reeth, K., Nauwynck, H., Pensaert, M., 2000. A potential role for tumour necrosis
factor- in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in
the induction of respiratory disease in pigs, J. Med. Microbiol. 49, 613–620.
Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A., Haesebrouck, F.,
2003. Evaluation of virulence of Mycoplasma hyopneumoniae field isolates, Vet. Microbiol.
97, 177–190.
Vicca, J., Maes, D., Jonker, L., De Kruif, A., Haesebrouck, F., 2005. The efficacy of tylosin
premix for the treatment and control of Mycoplasma hyopneumoniae infections. Vet. Rec.
156, 606-610.
Wise, K., and Kim, M., 1987. Major membrane surface proteins of Mycoplasma
hyopneumoniae selectively modified by covalently bound lipid. J. Bacteriol. 169, 5546–5555.
Whittlestone, P., 1972. The role of mycoplasmas in the production of pneumonia in the pig.
In: Pathogenic Mycoplasmas. Amsterdam. Associated Scientific Publishers. p. 263-283.
Young, T.F., Thacker, E.L., Erickson, Z., Ross, R.F., 2000. A tissue culture system to study
respiratory ciliary epithelial adherence of selected swine mycoplasmas. Vet. Microbiol.. 71:
269-79.
Zielinski, G., and Ross, R.F., 1992. Morphologic features and hydrophobicity of the cell
surface of Mycoplasma hyopneumoniae, Am. J. Vet. Res. 53, 1119–1124.
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CHAPTER 4.
GENERAL DISCUSSION
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GENERAL DISCUSSION
M. hyopneumoniae is the causal agent of enzootic pneumonia in pigs and represents a costly
problem for the pig industry worldwide. The clinical manifestation of the disease is the result
of a combination of farm management practices, environmental factors, secondary infections
and the virulence of the M. hyopneumoniae strains present in a herd (Vicca et al., 2003).
Notwithstanding the efforts and control strategies adopted to rear high health pig herds, M.
hyopneumoniae continues to be a widely spread organism in pig populations, which renders
eradication of this respiratory disease a very difficult and frustrating task. The present thesis
aimed to obtain a better epidemiological insight into early infections with M. hyopneumoniae
in herds with respiratory problems and the transmission in nursery pigs under field conditions.
Furthermore, the protection against experimental infection with different strains of M.
hyopneumoniae was investigated. On the one hand, the potential protection offered by a low
virulent strain was assessed. On the other hand, the efficacy of a currently existing vaccine
against infections with a low and a highly virulent strain was investigated. The present
chapter will provide a general discussion of the findings obtained in the different studies.
Epidemiology of M. hyopneumoniae infections in young pigs from herds with respiratory
problems
M. hyopneumoniae infections starting early in the production system play a critical role in
later development of respiratory disease in a pig herd (Fano et al., 2007). Although clinical
symptoms are not common before 6 weeks of age (Ross et al., 1999), it is known that the
lower and upper respiratory tract of piglets can already be colonized by M. hyopneumoniae
before weaning (Sibila et al., 2007; Moorkamp et al., 2009). These authors suggested that
prevalence at weaning can be used to predict the clinical course and severity of the disease
later on during the fattening period. Thus, to have a better understanding of the infection
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dynamics of this pathogen, it is important to assess the infection level at an early age. In our
first study (3.1), the infection level was estimated in piglets of 3 weeks of age in different
types of pig herds with respiratory problems in grow-finish pigs located in nine different
European countries. Of the total 52 pig herds included in this study, 28.6% were farrow-tofinish herds, 26.5% and 24.5% were sow herds from which the piglets were moved to another
farm after weaning or at 20-25 kg, respectively, and the remainder of the herds (20.4%) raised
a part of the fattening pigs on the same site.
The total average percentage of positive piglets was 10.7%. The results also showed that in
two thirds of the studied herds, there was evidence of M. hyopneumoniae infection in 3-week
old piglets. In herds vaccinating against M. hyopneumoniae 8.4% (95% CI 7.6-9.1) were
infected, whereas only 2.6% (95% CI 1.8-3.4) were infected in the herds not vaccinating
against M. hyopneumoniae. In most of the positive herds, the infection rate varied between 1
and 10%, whereas in some others more than 30% of the piglets were already positive.
The prevalence of M. hyopneumoniae in suckling piglets under field conditions has been
documented in other studies, but these studies were conducted in a limited number of herds
and in a particular geographic region (Sibila et al., 2007; Moorkamp et al., 2009; Nathues et
al., 2010). Yet, the average detection rate of 10.7% in suckling pigs estimated by our study
fits within the range of observations presented by other authors, considering that many of
them also included herds with a history of endemic respiratory disease. For instance,
Calsamiglia and Pijoan (2000) reported between 7.7% - 9.6% of pigs infected by 17 days of
age. Sibila et al. (2007) showed that the percentage of suckling pigs with M. hyopneumoniae
detection in nasal swabs was 1.5% - 3.8%. Nathues et al. (2010) observed an infection rate of
2.0%, while another study reported a higher prevalence of 12.3% in piglets of the same age
range (Moorkamp et al., 2009). Only one author reported a very wide range of infection (2.5 51.8%) in piglets from different batches all belonging to a multi-site farm, one day before
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weaning (Fano et al., 2006). However, such large variability could be due to the small number
of pigs sampled in each batch. Our study was the first to investigate the occurrence of this
pathogen in different countries in Europe and the results showed that in almost all countries
and type of herds, early M. hyopneumoniae infections are important.
A limited number of herds were included in the study; thus, the interpretation of these results
should be confined to herds encompassing the selection criteria, instead of being extrapolated
to the total population of pig herds within Europe. Generalization of these data might either
underestimate or overestimate the true prevalence of M. hyopneumoniae in suckling piglets
under field conditions.
It has been found that the prevalence of M. hyopneumoniae in young piglets may be
associated with different factors such as one-site and two-site production, the presence of
other respiratory pathogens in the herd (PRRSV, swine influenza virus, Pasteurella.
multocida, Haemophilus parasuis, Mycoplasma hyorhinis, Actinobacillus pleuropneumoniae
and Streptococcus suis) and inappropriate acclimatization of replacement gilts (Moorkamp et
al., 2009). In the final multivariable model of our study, it was shown that early M.
hyopneumoniae infection was correlated to herds practicing sow vaccination against swine
influenza virus (SIV). The exact reason for the higher risk of infection in herds vaccinating
their sows against SIV is not known. A possible explanation may be that pig herds with
respiratory symptoms are more likely to vaccinate their sows against SIV, overlooking the
possible influence of other respiratory pathogens such as M. hyopneumoniae. As most of the
respiratory problems are due to multiple infections and also to non-infectious factors (Thacker
et al., 2002), it is possible that in these herds, M. hyopneumoniae and/or other pathogens are
involved in respiratory problems in addition to SIV.
Apart from the sows playing a key role in the infection of their offspring, there are other
factors that may influence infection at a young age. For instance, piglets from different sows
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are often grouped together shortly after birth and later at weaning. Separation from the dam,
change of feed, housing, as well as the social stress inflicted by the effect of mixing, hierarchy
competition and dominance status during this period may suppress their immune function and
make them more vulnerable to infection (De Groot et al., 2001). As a consequence, M.
hyopneumoniae organisms continue to spread horizontally between pen mates in the nursery
unit (Sibila et al., 2009). Moreover, airborne transmission can also occur, leading to
transmission of M. hyopneumoniae between pigs of different units within a herd. The
detection rate of M. hyopneumoniae in the piglets was not significantly different between
herds practicing vaccination and those not practicing vaccination. The absence of a difference
in the present study can be due to differences in vaccination strategies employed by the herds
(e.g. type of vaccine, timing of vaccination, etc). Piglet vaccination constitutes one of the
main strategies currently used for control of M. hyopneumoniae infection. Therefore, it is
important to assess whether the vaccination of weaned piglets influences the extent of M.
hyopneumoniae transmission during the nursery period.
Transmission of M. hyopneumoniae in vaccinated and non-vaccinated nursery pigs
Our second study (3.2) aimed to quantify M. hyopneumoniae transmission in nursery pigs
under field conditions, and to assess the effect of vaccination on transmission. Bronchoalveolar lavage fluid was collected at weaning and at the end of the nursery period to assess
the presence of M. hyopneumoniae by nPCR and to calculate the reproduction ratio (Rn). The
average Rn- values obtained were 0.56 and 0.71 for the non vaccinated and the vaccinated
group, respectively, indicating that vaccination alone was not able to significantly reduce the
transmission of this pathogen in nursery pigs in the present herd.
An earlier trial under experimental conditions (Meyns et al., 2006) showed similar findings
and demonstrated that vaccination with another conventional vaccine could not prevent
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infection nor transmission of this organism. The average Rn- values obtained in our study
were lower than those obtained under experimental conditions (Meyns et al., 2004, 2006),
which could be influenced, among other factors, by the infection dose. The infection dose in
the present study is not known, but it is expected that the dose used in the previous
experimental trials (Meyns et al., 2004; 2006) was higher than the infection dose under field
conditions at weaning. A lower infection dose may result in a lower number of M.
hyopneumoniae organisms in the respiratory tract, a lower transmission rate, and
consequently, a lower Rn value.
Despite the lack of significance in the difference of the transmission rate between vaccinated
and non vaccinated animals, some observations suggest that the load of M. hyopneumoniae in
the lungs of infected animals may be influenced by vaccination. For instance, Meyns et al.
(2006) used an IF-staining method to measure in a semi-quantitative way the number of M.
hyopneumoniae bacteria in the lung tissue of pigs. They found that the lung samples of
vaccinated animals had a lower average IF score than the lungs of non vaccinated ones,
possibly also resulting in lower excretion.
The virulence of the M. hyopneumoniae strain might influence the number of organisms
present in the respiratory tract of an infected pig. Although Meyns et al. (2004) did not find
statistically significant differences between transmission rate of a highly and a low virulent
strain, it was shown that with the same infection dose, the lung tissue of pigs infected with a
highly virulent strain displayed a more intense IF-staining than the lung tissue of pigs infected
with a low virulent strain (Vicca et al., 2003; Meyns et al., 2004). In addition, the titer of M.
hyopneumoniae was significantly higher in the BAL fluids of the pigs infected with a highly
virulent strain at 10 and 15 DPI compared with the titer in BAL fluid of pigs infected with the
low virulent strain (Meyns et al., 2007). It was suggested that, among other factors, the faster
in vivo multiplying capacity of the highly virulent strain could explain the higher titers found
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in the lungs. The virulence of the M. hyopneumoniae strain in our field study was not known,
and also, it was not known whether the pigs were infected with only one or with different M.
hyopneumoniae strains. Previous research has shown that diversity among M. hyopneumoniae
strains circulating in pig herds may occur (Vicca et al., 2003; Calus et al., 2007). Further
research however is necessary to assess the importance of this diversity from a clinical and
epidemiological point of view, and ultimately, for the implementation of optimal control
strategies.
In our study, piglets were born from sows vaccinated against M. hyopneumoniae at 5 and 3
weeks prior to farrowing. The effect of sow vaccination has been documented by several
studies (Ruiz et al., 2003, Kristensen et al., 2004, Beilage et al., 2005, Martelli et al., 2006,
Sibila et al., 2008), and it is known to enhance the levels of maternally derived antibodies
during the first weeks of life of the offspring. At the same time, in some studies, it seems to be
able to reduce prevalence of M. hyopneumoniae in piglets at weaning at the herd level (Ruiz
et al., 2003; Beilage et al., 2005). Thacker et al. (2000) however observed only limited to no
effect on respiratory tract colonisation by this pathogen.
It is not clear whether high levels of maternally derived antibodies may interfere with early
(one-shot) vaccination (Beilage et al., 2005). According to a study by Martelli et al. (2006),
sow vaccination did not have an effect on the serological response of vaccinated piglets. It is
difficult, therefore, to say whether the higher level of antibodies in the vaccinated than in the
non-vaccinated group at the start of this study may have interfered with the efficacy of
vaccination at 3 weeks of age, and possibly influenced the transmission ratio among piglets.
More research on the influence of maternally derived antibodies on the immunization of
piglets, and the best timing for vaccination, is necessary.
The Rn calculations in this study were based on the number of pigs infected at the end of the
nursery period. To measure the infectious state of the animals, nPCR was performed on BAL
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fluid obtained from live anesthesized animals. Since M. hyopneumoniae attaches to the
ciliated epithelium of the respiratory tract, the best samples to detect this organism by nPCR
are tracheo-bronchial swabs or BAL fluid (Marois et al., 2008; Fablet et al., 2010). BAL fluid
collection can be time and labour-expensive, but the high specificity and sensitivity of nPCR
on this type of sample offers several advantages over other diagnostic methods. Its sensitivity
enables the detection of 5.106 ng M. hyopneumoniae DNA or as few as 4 organisms / μl
reaction mixture (Gebruers et al., 2008). For practical reasons, other studies have used nPCR
on nasal swabs to determine prevalence in young piglets (Sibila et al., 2007). However, the
number of organisms in the upper respiratory tract is lower and organisms are shed
intermittently (Kurth et al., 2002; Ruiz et al., 2002; Pieters et al., 2009). In addition, inhibiting
factors such as mucin can also interfere in the DNA extraction and affect the process of
amplification (Vranckx K. 2010, personal communication). Nonetheless, nPCR performed on
nasal swabs is considered to be appropriate to assess the presence of M. hyopneumoniae at
herd level, but not to assess the infection at the animal level (Otagiri et al., 2005; Sibila et al.,
2009).
Until recently, no accurate diagnostic tools were available for quantifying the bacterial load of
this organism in the BAL fluid from live naturally infected animals. However, new methods
have recently been developed to increase accuracy in the measurements. The best quantitative
technique available nowadays is the real-time PCR assay for M. hyopneumoniae (Marois et
al., 2010; Fablet et al., 2010). Real-time PCR is an accurate, fast and easy to perform
technique that offers new possibilities for transmission studies, taking epidemiological
research from qualitative identification to precise quantification of this organism. Although
this tool was still under development during the performance of our study, it should certainly
be considered in future studies investigating the effect of vaccination on the possible
reduction of the load of this microorganism in the respiratory tract of infected pigs. The
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quantification of the number of organisms being shed by an animal per unit of time would
provide highly valuable information in M. hyopneumoniae research and could reveal new
insights in the transmission of this pathogen.
It is important to point out that the Rn- values in this study correspond only to the nursery
population. An Rn- value lower than one would mean lack of transmission only at that stage,
but not in the whole pig population, since infection could be already established at an earlier
age (chapter 3.1) or could occur at a later stage. Thus, eradication of M. hyopneumoniae will
only be achieved when appropriate control measures will lower the average Rn- value to less
than one for all circulating M. hyopneumoniae strains, at all stages in the pig population. To
achieve such results, a vaccine would either need to decrease the infectivity of animals that
spread the pathogen and/or induce better resistance in animals that are still susceptible to
infection. So, additional control measures and/or more efficient vaccines will be needed to
achieve this goal.
Infection pattern of M. hyopneumoniae strains of different virulence in vaccinated and nonvaccinated pigs
From our study (3.2) and previous experimental trials (Meyns et al., 2006; Pieters et al.,
2010), it is clear that the current commercial vaccines are not able to stop transmission.
However, little is known about the effect of vaccination against strains of different virulence.
Strait et al. (2008) suggested that differences in virulence may affect the ability of current
vaccines to protect against different M. hyopneumoniae field isolates. Our third study (3.3)
aimed to investigate the infection pattern and the lung lesion development caused by a low
and a highly virulent strain, and to examine the efficacy of vaccination against each one of
them.
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This study confirmed the difference in virulence between these two strains both at 4 weeks PI
and at 8 weeks PI. In the commonly accepted standard infection model (Whittlestone et al.,
1972; Kobisch et al., 1993) used in previous experiments, clinical signs and lung lesions are
assessed up to 4 weeks PI. Our study was the first experimental trial to examine the pattern of
infection of both strains over a period of 8 weeks PI.
It was demonstrated that, contrary to the highly virulent strain, the low virulent strain requires
more than 4 weeks PI to reach maximum symptoms. Apart from developing milder
symptoms, the onset of disease in pigs infected with the low virulent strain only started at
about 4 weeks PI, whereas at this point in time symptoms were already at their maximum in
pigs infected with the highly virulent strain. This is a clear indication that the standard
infection model is not entirely suitable for the evaluation of the course of disease caused by
low virulent strains. For these M. hyopneumoniae strains, it is necessary to observe the pigs
for a longer period than the traditional 4 weeks period. Further research is necessary to
investigate why the infection and disease course is different between low and highly virulent
strains.
In addition to the differences in virulence and effect of time on the infection course, the
efficacy of vaccination against highly and low virulent strains was also evaluated. It was
observed that vaccination delayed the clinical onset of disease and significantly reduced its
severity, both in pigs infected with the highly virulent strain and in those infected with the
low virulent strain. Disease reduction by vaccination, however, was more prominent in pigs
challenged with the highly virulent strain than in pigs challenged with the low virulent strain.
Part of the different effects might be explained by the fact that significant vaccination efficacy
is more visible when clinical symptoms and lung lesions are more severe, leaving more room
for improvement by vaccination. Another possibility is that vaccination may affect differently
the multiplication rate of M. hyopneumoniae in the lungs for the highly and low virulent
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strain. Vaccination significantly reduced IF scores in the pigs infected with the highly virulent
strain, but not in pigs infected with the low virulent strain. This might suggest that vaccination
inhibits the multiplication of the highly virulent strain to a higher extent compared to the low
virulent one. It would be interesting to quantify more accurately the effect of vaccination on
the load of organisms in the lungs, for instance by using real-time PCR.
To better understand the effect of vaccination on multiplication of different strains in the
respiratory tract, more information is also required on the immunological response of the host.
The existing reports on immunity induced by vaccination suggest that both cellular and
humoral responses are involved in protection (Chen et al., 2008; Strait et al., 2008). Strains
with different virulence might trigger mechanisms of the immune response to a different
extent (Meyns et al., 2007). In fact, a higher proportion of neutrophils in BAL fluid and
higher level of TNF-α have been reported for non vaccinated pigs infected with the highly
virulent strain, indicating that the inflammatory cell response is more pronounced in case of
infections with the highly virulent strain (Meyns et al., 2007). Whether this also occurs in
vaccinated pigs and whether this affects vaccine efficacy may be the subject of further studies.
Since infected pigs are often purchased and transferred from one herd to another, it can be
expected that different strains may be circulating under field conditions (Mayor et al., 2007).
A high heterogeneity at the genomic and proteomic level has been demonstrated among
different M. hyopneumoniae strains (Artiushin and Minion, 1996; Kokotovic et al., 1999;
Stakenborg et al., 2005; Calus et al., 2007). Some of these variations among field strains could
be held responsible for the variable outcome of vaccination strategies in different herds (Maes
et al., 1999). Also, a high variation in virulence exists between M. hyopneumoniae strains
isolated from different swine herds (Vicca et al., 2003). However, the virulence factors of M.
hyopneumoniae have not been identified and the protective antigens remain undefined. The
importance of virulence differences under field conditions for protective immunity by
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vaccination is unknown. Further research is necessary to investigate the role of diversity
between strains under field conditions and cross-protection between a commercial vaccine
strain and the most prevalent strains in a herd. A possible future for the development of
alternative vaccines may lie in the identification of new, preferably protective, antigens from
different strains. These antigens could then be included in the vaccine, allowing a broader
antigenic coverage against different M. hyopneumoniae strains.
Adjuvants may also play an important role in the enhancement of the immunogenicity of an
antigen. In the case of commercial vaccines against M. hyopneumoniae, most of the
formulations contain oil-based adjuvants (usually in the form of emulsions), giving a more
prolonged release of the antigen and stimulation of the immune system (Groth et al., 2001).
An adjuvant consisting of a mixture of amphigen and lecithin was present in the vaccine
formulation used in our study (3.3). Although the currently used bacterin formulations are
able to reduce lung lesions and improve performance parameters of pigs affected by M.
hyopneumoniae, the advantages of different adjuvants are still under debate. More studies
could elucidate the protective effect of different vaccine formulations against infection with
different M. hyopneumoniae strains.
Combined infections with different M. hyopneumoniae strains
The effect of combined infections with different M. hyopneumoniae strains had previously
been reported in only one study (Strait et al., 2008). These authors investigated the effect of
vaccination on the infection with two contemporary pathogenic isolates of M.
hyopneumoniae. However, the possible protective effect offered by infection with one strain
against subsequent infection with another strain was not investigated. Therefore, in chapter
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3.4, we investigated the effect of infection of pigs with two different low virulent strains (F1
and F13) on a subsequent challenge infection with a highly virulent (F7) strain.
The results clearly showed that the low virulent strains were not able to induce protection
against the highly virulent strain. On the contrary, the severity of the clinical parameters,
macroscopic, histopathological lesions and IF scores was exacerbated in pigs that had been
first infected with a low virulent strain. This suggests that although low virulent strains do not
induce severe clinical symptoms by themselves, they may play an important role in increasing
the severity of subsequent infection with a highly virulent isolate. These results were
surprising, since we expected the low virulent strain to confer at least a certain degree of
protection against the highly virulent strain. Contrary to our study, Kobisch et al. (1993)
found in a limited study that resistance to pneumonia was observed in pigs after a booster
infection with the same isolate of M. hyopneumoniae. The exact reason for the lack of
protection of the low strains against the highly virulent strain in our study is not known, but it
might be that the host immune system responds with a more pronounced inflammatory
reaction when subsequently exposed to more than one strain. The route of inoculation
(endotracheal), being a rather invasive procedure, could also have contributed a more
pronounced inflammatory response. For instance, it has been demonstrated that animals
receiving particles through inhalation showed a decreased pulmonary response, in both
severity and persistence, when compared with those receiving particles through instillation
(Osier et al., 1997). The differences in pulmonary response when using alternative routes of
infection may be caused by differences in dose rate, particle distribution, or altered clearance.
Compared to natural infection (under field conditions), the inoculation dose with each one of
the strains is likely to be higher under the experimental conditions. This might result in a
faster and a higher level of colonization of the ciliated surface of the respiratory tract by M.
hyopneumoniae organisms, triggering more pronounced symptoms and lesions.
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CHAPTER 4: DISCUSSION
Another possibility could be that the low virulent strains require more than 4 weeks to induce
protection. Therefore, the time between the first and the second challenge could have been
insufficient to trigger a full protective immune reaction in the host. A short-time interval
between subsequent exposures of different strains may leave a window of susceptibility that
can result in super-infection with exacerbated clinical signs and lung lesions. Superinfection
is defined as infection with a second strain after the establishment of persistent infection and
development of an immunologic response to the first strain (Blackard et al. 2007). The
importance of time interval between challenge exposure is not well documented for M.
hyopneumoniae, but it is a factor to be considered in future experiments. More knowledge
about the immunological response in the animal following M. hyopneumoniae infection with
individual strains, as well as the optimal time between exposures are necessary for a better
understanding of the interaction between strains of different virulence.
As the results did not give any indications of a protective immunity induced by infection with
the low virulent strains on subsequent infection with a highly virulent strain, the low virulent
strains used in this study should be regarded cautiously as candidates for vaccines. Given the
time interval between primary and subsequent exposure, the results also imply that infection
with more than one M. hyopneumoniae strain may lead to more severe clinical disease in a pig
herd.
General conclusions
From this thesis, it can be concluded that:

M. hyopneumoniae infections in 3-week old piglets commonly occur in European pig
herds suffering from respiratory disease in grow-finishing pigs.

Vaccination against M. hyopneumoniae is not able to significantly reduce the
transmission of this pathogen in nursery pigs under field conditions.
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CHAPTER 4: DISCUSSION

Infection with a low virulent M. hyopneumoniae strain requires more than 4 weeks to
reach maximum symptoms, indicating that the standard M. hyopneumoniae infection
model based on a 4-week observation period is not entirely suitable to evaluate low
virulent strains.

A commercial bacterin seems to be more efficacious against infection with the highly
virulent M. hyopneumoniae strain than with the low virulent one used in this study.

Previous infection with the low virulent M. hyopneumoniae strains does not confer
protection against subsequent infection with the highly virulent strain used in this
study.
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CHAPTER 4: DISCUSSION
Future perspectives
From the literature review and this discussion, it appears that still many questions on M.
hyopneumoniae infections remain unanswered. Therefore, further research to answer these
questions and to achieve a better and more sustainable control of enzootic pneumonia is
necessary.
The prevalence of M. hyopneumoniae infections in young piglets at weaning has been
addressed, and possible risk factors have been investigated. The sow is known to play an
important role in the transmission of this pathogen to her offspring. However, the
transmission rate of this pathogen from sows to piglets has not yet been quantified, and the
difference between young and older sows in transmission of M. hyopneumoniae to the
offspring is not known. Also, factors influencing the occurrence and transmission of M.
hyopneumoniae infections in sows are largely unknown. These may include housing
conditions and management practice, parity and vaccination schemes in the herd, amongst
others.
No significant difference in transmission rate was observed between piglets vaccinated
against M. hyopneumoniae and non vaccinated ones. However, it is not known whether
vaccination can reduce the load of M. hyopneumoniae bacteria in the lungs of infected piglets.
Thus, future studies should quantify more accurately e.g. by using qPCR, the number of M.
hyopneumoniae bacteria in the lungs and should investigate whether control measures are able
to significantly decrease the infection load in the lungs.
Finally, the interactions between highly and low virulent strains need to be better understood.
In our studies, pigs subsequently infected with different strains developed more severe
clinical symptoms and lesions. The clinical importance of this finding in pig herds is not
known. It is, yet, to be found whether the time interval between challenge exposures plays a
193
CHAPTER 4: DISCUSSION
role in the degree of protection conferred. It is also not known whether simultaneous infection
with two different strains will lead to more severe disease, and whether both strains will
multiply at the same rate in the pig, or whether one strain will dominate the other one. The
interactions between M. hyopneumoniae and other respiratory pathogens also need to be
better understood.
These are important questions as they can help explaining the clinical course of the infection,
the persistence of infection in pigs and the chronic nature of the disease in pig herds. Also the
effect of vaccination against simultaneous infection with different strains should be
investigated, as this may help explaining the variable vaccination results observed in pig
herds.
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CHAPTER 4: DISCUSSION
References
Artiushin, S., Minion, F.C., 1996. Arbitrarily primed PCR analysis of Mycoplasma
hyopneumoniae field isolates demonstrates genetic heterogeneity. Int. J. Syst. Bacteriol. 46,
324-328.
Calsamiglia, M., Collins, J.E., Pijoan, C., 2000. Correlation between the presence of enzootic
pneumonia lesions and detection of Mycoplasma hyopneumoniae in bronchial swabs by PCR.
Vet. Microbiol. 76, 299-303.
Calsamiglia, M., Pijoan, C., 2000. Colonisation state and colostral immunity to Mycoplasma
hyopneumoniae of different parity sows. Vet. Rec. 146, 530-532.
Calus, D., Baele, M., Meyns, T., de, K.A., Butaye, P., Decostere, A., Haesebrouck, F., Maes,
D., 2007. Protein variability among Mycoplasma hyopneumoniae isolates. Vet. Microbiol.
120, 284-291.
Chen, A.Y., Fry, S.R., Daggard, G.E., Mukkur, T.K., 2008. Evaluation of immune response to
recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as
cocktail DNA and/or recombinant protein vaccines in mice. Vaccine 26, 4372-4378.
de Groot, J., Ruis, M.A., Scholten, J.W., Koolhaas, J.M., Boersma, W.J., 2001. Long-term
effects of social stress on antiviral immunity in pigs. Physiol. Behav. 73, 145-158.
Dunn, R. J., Witter, R., Silva, R., Lucy F. LeeA, Finlay, J., Marker, B., Kaneene, J., Fulton,
R., Fitzgerald, S., 2010. The Effect of the Time Interval Between Exposures on the
Susceptibility of Chickens to Superinfection with Marek's Disease Virus. Avian Diseases 54,
1038-1049.
Fablet, C., Marois, C., Kobisch, M., Madec, F., Rose, N., 2010. Estimation of the sensitivity
of four sampling methods for Mycoplasma hyopneumoniae detection in live pigs using a
Bayesian approach. Vet. Microbiol. 143, 238-245.
Fano, E., Pijoan, C., Dee, S., Deen, J., 2007. Effect of Mycoplasma hyopneumoniae
colonization at weaning on disease severity in growing pigs. Can. J. Vet. Res. 71, 195-200.
195
CHAPTER 4: DISCUSSION
Gebruers, F., Calus, D., Maes, D., Villarreal, I., Pasmans, F., Haesebrouck, F., 2008. A set of
four nested PCRs to detect different strains of Mycoplasma hyopneumoniae in biological
samples. In: Proceedings of the 17th Congress of the International Organization for
Mycoplasmology, Tianjin, China, pp. 90.
grosse Beilage, E., Schreiber, A., 2005. Impfung von Sauen gegen Mycoplasma
hyopneumoniae mit Hyoresp®/ Vaccination of sows against Mycoplasma hyopneumoniae
with Hyoresp®. Dtsch. Tierarztl. Wochenschr. 112, 256-261.
Groth, D., Rapp-Gabrielson, V. , 2001. Evaluation of M+PAC in one and two dose-regimens
against competitor one-dose Mycoplasma hyopneumoniae bacterins. Proceedings of the Allan
D. Leman Swine Conference, Vol. 38, Supplement, University of Minnesota, USA, p41.
Kobisch, M., Blanchard, B., Le Potier, M.F., 1993. Mycoplasma hyopneumoniae infection in
pigs: duration of the disease and resistance to reinfection. Vet. Res. 24, 67-77.
Kokotovic, B., Friis, N.F., Jensen, J.S., Ahrens, P., 1999. Amplified-fragment length
polymorphism fingerprinting of Mycoplasma species. J. Clin. Microbiol. 37, 3300-3307.
Kristensen, C., Andreasen, M., Ersbøll, A., and Nielsen, J. P., 2004. Antibody response in
sows and piglets following vaccination against Mycoplasma hyopneumoniae, toxigenic
Pasteurella multocida, and Actinobacillus pleuropneumoniae. Can. J. Vet. Res. 68, 66–70.
Kurth, K.T., Hsu, T., Snook, E.R., Thacker, E.L., Thacker, B.J., Minion, F.C., 2002. Use of a
Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal
sampling sites in swine. J. Vet. Diagn. Invest. 14, 463-469.
Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., de, K.A., 1999.
Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine influenza
viruses and Aujeszky's disease virus in slaughter pigs from fattening pig herds. Zentralbl.
Veterinarmed. B 46, 341-352.
Marois, D., Dory, D., Fable, C., Madec, F., Kobisch, M., 2010. Development of a quantitative
Real-Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma
hyopneumoniae strain 116 required to induce pneumonia in SPF pigs. J. Appl. Microbiol. 108,
1523-1533.
196
CHAPTER 4: DISCUSSION
Martelli, P., Terreni, M., Guazzetti, S., Cavirani, S., 2006. Antibody response to Mycoplasma
hyopneumoniae infection in vaccinated pigs with or without maternal antibodies induced by
sow vaccination. J. Vet. Med. B Infect. Dis. Vet. Public Health 53, 229-233.
Mayor, D., Zeeh, F., Frey, J., Kuhnert, P., 2007. Diversity of Mycoplasma hyopneumoniae in
pig farms revealed by direct molecular typing of clinical material. Vet. Res. 38, 391-398.
Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F., de, K.A., 2004. Quantification of
the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments.
Prev. Vet. Med. 66, 265-275.
Meyns, T., Dewulf, J., de, K.A., Calus, D., Haesebrouck, F., Maes, D., 2006. Comparison of
transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations.
Vaccine 24, 7081-7086.
Meyns, T., Maes, D., Calus, D., Ribbens, S., Dewulf, J., Chiers, K., de, K.A., Cox, E.,
Decostere, A., Haesebrouck, F., 2007. Interactions of highly and low virulent Mycoplasma
hyopneumoniae isolates with the respiratory tract of pigs. Vet. Microbiol. 120, 87-95.
Moorkamp, L., Hewicker-Trautwein, M., grosse Beilage, E., 2009. Occurrence of
Mycoplasma hyopneumoniae in coughing piglets (3-6 weeks of age) from 50 herds with a
history of endemic respiratory disease. Transbound. Emerg. Dis. 56, 54-56.
Nathues, H., Strutzberg-Minder, K., Kreienbrock, L., grosse Beilage, E., 2010. Occurrence of
Mycoplasma hyopneumoniae infections in suckling and nursery pigs in a region of high pig
density. Vet. Rec. 13, 194-198.
Osier, M., Oberörster, G., 1997. Intratracheal Inhalation vs Intratracheal Instillation:
Differences in Particle Effects. Toxicol. Sci. 40, 220-227.
Otagiri, Y., Asai, T., Okada, M., Uto, T., Yazawa, S., Hirai, H., Shibata, I., Sato, S., 2005.
Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by
nested PCR and culture methods. J. Vet. Med. Sci. 67, 801-805.
Pieters, M., Pijoan, C., Fano, E., Dee, S., 2009. An assessment of the duration of Mycoplasma
hyopneumoniae infection in an experimentally infected population of pigs. Vet. Microbiol.
134, 261-266.
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Pieters, M., Fano, E., Pijoan,
C., Dee, S., 2010. An experimental model to evaluate
Mycoplasma hyopneumoniae transmission from asymptomatic carriers to unvaccinated and
vaccinated sentinel pigs. Can. J. Vet. Res. 74, 157–160.
Ross, R., 1999. Mycoplasmal diseases. In: Leman, A.D., Straw, B.E., D‘Allaire, S.,
Mengeling, W.L., and Taylor, D.J., (Eds), Diseases of Swine, 8th edn. The Iowa State
University Press, Ames, IA, pp 495-509.
Ruiz, A., Galina, L., Pijoan, C., 2002. Mycoplasma hyopneumoniae colonization of pigs sired
by different boars. Can. J. Vet. Res. 66, 79-85.
Ruiz, A., Utrera, V., Pijoan, C., 2003. Effect of Mycoplasma hyopneumoniae sow vaccination
on piglet colonization at weaning. J. Swine Health Prod. 11, 131-135.
Sibila, M., Nofrarias, M., Lopez-Soria, S., Segales, J., Riera, P., Llopart, D., Calsamiglia, M.,
2007. Exploratory field study on Mycoplasma hyopneumoniae infection in suckling pigs. Vet.
Microbiol. 121, 352-356.
Sibila, M., Bernal, R., Torrents, D., Riera, P., Llopart, D., Calsamiglia, M., Segales, J., 2008.
Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet
colonization and seroconversion, and pig lung lesions at slaughter. Vet. Microbiol. 127, 165170.
Sibila, M., Pieters, M., Molitor, T., Maes, D., Haesebrouck, F., Segales, J., 2009. Current
perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection.
Vet. J. 181, 221-231.
Simionatto, S., Marchioro, S.B., Galli, V., Luerce, T.D., Hartwig, D.D., Moreira, A.N.,
Dellagostin, O.A., 2009. Efficient site-directed mutagenesis using an overlap extension-PCR
method for expressing Mycoplasma hyopneumoniae genes in Escherichia coli. J. Microbiol.
Methods.
Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Minion, F.C., Peeters, J., de, K.A.,
Haesebrouck, F., 2005. Characterization of In Vivo acquired resistance of Mycoplasma
hyopneumoniae to macrolides and lincosamides. Microb. Drug Resist. 11, 290-294.
198
CHAPTER 4: DISCUSSION
Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Peeters, J., de, K.A., Haesebrouck, F., 2005.
The diversity of Mycoplasma hyopneumoniae within and between herds using pulsed-field gel
electrophoresis. Vet. Microbiol. 109, 29-36.
Strait, E.L., Madsen, M.L., Minion, F.C., Christopher-Hennings, J., Dammen, M., Jones,
K.R., Thacker, E.L., 2008. Real-time PCR assays to address genetic diversity among strains
of Mycoplasma hyopneumoniae. J. Clin. Microbiol. 46, 2491-2498.
Thacker B., Thacker E., Halbur P., Minion C., Young T., Erickson B.,Thanawonguwech T.,
2000. The influence of maternally-derived antibodies on Mycoplasma hyopneumoniae
infection. In: Proc 16th IPVS congress, Melbourne Australia, 454.
Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de, K.A., Haesebrouck, F., 2003.
Evaluation of virulence of Mycoplasma hyopneumoniae field isolates. Vet. Microbiol. 97,
177-190.
Whittlestone, P., 1972. The role of mycoplasmas in the production of pneumonia in the pig.
In: Pathogenic Mycoplasmas. Associated Scientific Publishers, Amsterdam, 263-283.
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SUMMARY
M. hyopneumoniae is the primary agent of enzootic pneumonia, a chronic respiratory disease
in pigs that occurs worldwide and causes major economic losses. The clinical course of
infections may vary significantly between pig herds depending on herd management
strategies, secondary infections and environmental conditions. Also the virulence of the M.
hyopneumoniae strains may determine the clinical outcome, which renders control of this
pathogen a difficult task. Control measures include the optimization of management and
housing conditions, the use of strategic medication and/or vaccination. The current control
measures are beneficial from an economic point of view, but they do not entail a sustainable
solution for the disease.
There is limited information on the occurrence and risk factors involved in early infection and
there are still many questions regarding the effect of vaccination on transmission of this
pathogen. The limited studies available have mainly focused on experimental conditions and
do not necessarily reflect the situation in the field. In addition, information on differences
between infection course caused by low and highly virulent field strains and the effect of
vaccination on infection with different M. hyopneumoniae strains is very scarce. Answers to
these questions are necessary for a better understanding of the pathogenicity of this
microorganism, and to improve the control of enzootic pneumonia.
The general aim of this study was to assess the importance of early infection and transmission
of M. hyopneumoniae under field conditions and to investigate protection against different M.
hyopneumoniae strains.
In the first study (Chapter 3.1), the detection rate of M. hyopneumoniae in 3-week-old pigs in
different European countries was estimated and possible risk factors were identified. Nasal
swabs from suckling pigs in 52 farms were collected for analysis using nested PCR. Potential
risk factors for respiratory disease were analysed with a multivariable logistic regression
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SUMMARY - SAMENVATTING
model. The average percentage of positive piglets was 10.7% (95% confidence interval, CI
7.4-14.2); at least one pig tested positive in 68% of herds. In 32% of the herds, more than
10% of piglets tested positive. Herds that vaccinated sows against swine influenza virus (SIV)
had a significantly higher risk of a piglet being positive for M. hyopneumoniae (OR 3.12;
95% CI 1.43-6.83). The higher risk in case of SIV vaccination is difficult to explain, but it
may be due to the fact that pig herds with respiratory symptoms are more likely to be
vaccinated against SIV, overlooking the possible influence of other respiratory pathogens
such as M. hyopneumoniae. Our results confirmed that M. hyopneumoniae is widespread in 3week-old piglets across different European countries.
Piglet vaccination constitutes one of the main strategies currently used for control of M.
hyopneumoniae infections. However, it is not known whether vaccination of weaned piglets
influences the extent of M. hyopneumoniae transmission under field conditions during the
nursery period. In Chapter 3.2, a study investigated the effect of vaccination against
Mycoplasma hyopneumoniae on its transmission in nursery pigs under field conditions.
Seventy two pigs were randomly allocated at weaning into vaccinated (V) and non-vaccinated
(NV) groups. Animals in the V group were vaccinated at 3 weeks-of-age with a commercial
M. hyopneumonaie bacterin vaccine. Bronchoalveolar lavage fluid taken at weaning and at the
end of the nursery period was assessed for the presence of M. hyopneumoniae by nested PCR,
and the reproduction ratio of infection (Rn) was calculated. The percentage of positive pigs in
the V and NV groups was 14% and 36% at weaning, and 31% and 64% at the end of the
nursery period, respectively. The Rn-values for the V and NV groups were 0.71 and 0.56,
respectively (P > 0.05). The study indicated that vaccination does not significantly reduce the
transmission of this respiratory pathogen.
In the third study (Chapter 3.3), the infection pattern and lung lesion development in pigs
caused by a low and highly virulent M. hyopneumoniae strain at 4 and 8 weeks (w) post
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SUMMARY - SAMENVATTING
infection (PI) was investigated. The efficacy of a commercial inactivated whole-cell vaccine
against infection with each one of these M. hyopneumoniae strains was also determined.
Ninety piglets free of M. hyopneumoniae were selected, and 40 of them were randomly
vaccinated during their first week of life. At weaning, all piglets were allocated to 10 different
groups and housed in pens with absolute filters. At 4 weeks of age, pigs were inoculated
intratracheally with either a highly virulent M. hyopneumoniae strain, a low virulent strain or
with sterile culture medium. Half of all animals were euthanized at 4w PI, while the
remaining half was euthanized at 8w PI. Coughing was assessed daily, and lung lesions,
immunofluorescence (IF), bacteriological analysis and nested PCR were assessed after
necropsy. It was demonstrated that contrary to the highly virulent strain, the low virulent
strain required more than 4 weeks PI (commonly accepted as the standard infection model) to
reach maximum clinical symptoms. Vaccination significantly reduced clinical symptoms,
macroscopic and microscopic lung lesions in pigs infected with the highly virulent strain. This
effect was more pronounced at 4 than at 8 weeks PI. Protective efficacy was also observed in
pigs infected with the low virulent strain, but the effect was less pronounced than on the
highly virulent strain.
Finally, the effect of an infection with low virulent isolates of M. hyopneumoniae (LV1 and
LV2) on the subsequent infection with a highly virulent isolate (HV) was evaluated (Chapter
3.4). Fifty-five, 3 week-old piglets free of M. hyopneumoniae were randomly allocated to 6
different groups. At 4 weeks of age (D0), groups LV1-HV and LV1 were intratracheally
inoculated with LV1, groups LV2-HV and LV2 with LV2, and group HV with sterile culture
medium. Four weeks later (D28), the pigs of these different groups were either intratracheally
inoculated with the highly virulent isolate (groups LV1-HV, LV2-HV, HV) or with sterile
culture medium (groups LV1 and LV2). A negative control group consisted of pigs inoculated
with sterile culture medium on D0 and D28. All animals were necropsied at 28 days after the
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SUMMARY - SAMENVATTING
second inoculation. Clinical symptoms were evaluated daily using a respiratory disease score
(RDS). After necropsy, macroscopic and histopathological lung lesions were quantified and
immunofluorescence (IF) testing on lung tissue and nested PCR on BAL fluid were
performed for the detection of M. hyopneumoniae. Disease signs and lung lesions were not
observed in pigs of the negative control group. In the other groups, there were no or only very
mild clinical symptoms from D0 until D28. A significant increase in the average RDS values
was, however, observed during D28-D56, especially in groups LV1-HV (1.48) and LV2-HV
(1.49), in group HV (0.79), and to a lesser extent in groups LV1 (0.50) and LV2 (0.65)
(P<0.05). The clinical symptoms during D28-D56, the lung lesions and intensity of IF
staining were more pronounced in groups LV1-HV, LV2-HV and HV compared to groups
LV1 and LV2. All pigs, except those from the negative control group, were positive on IF
testing and PCR at D56. The study demonstrated that pigs inoculated with low virulent
isolates of M. hyopneumoniae are not protected against a subsequent infection with a highly
virulent isolate 4 weeks later and may even develop more severe disease signs. This indicated
that subsequent infections with different M. hyopneumoniae isolates may lead to more severe
clinical disease in a pig herd.
From the studies included in this thesis, it was concluded that M. hyopneumoniae infections
start at an early age and are widely spread in European pig herds. Many questions remain still
unanswered and more detailed studies are necessary to assess the possible risk factors
associated to these infections.
No significant difference in transmission rate of M. hyopneumoniae was observed between
piglets vaccinated against M. hyopneumoniae and non vaccinated ones. This means that
vaccination alone will not be sufficient to significantly decrease M. hyopneumoniae infections
and that additional control measures will be needed for elimination of the pathogen from pig
herds. Future studies should quantify more accurately the number of M. hyopneumoniae
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SUMMARY - SAMENVATTING
bacteria in the lungs and should investigate whether control measures are able to significantly
decrease the infection load in the lungs.
The infection pattern and disease course following experimental infection differ between high
and low virulent M. hyopneumoniae strains, and also the efficacy of vaccination seems to be
determined by the type of strain. A better understanding of the infection pattern and the
interactions between highly and low virulent strains is necessary. Pigs subsequently infected
with different strains developed more severe clinical symptoms and lesions. However, it is not
known whether simultaneous infection with two different strains will lead to more severe
disease, and whether both strains will multiply at the same rate in the pig, or whether one
strain will dominate the other one. These are important questions as they can help explaining
the clinical course of the infection and improve the currently existing control measures
against enzootic pneumonia.
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SUMMARY - SAMENVATTING
SAMENVATTING
M. hyopneumoniae is het belangrijkste agens van enzoötische pneumonie, een chronische
luchtwegaandoening bij varkens die wereldwijd voorkomt en leidt tot grote economische
verliezen. Het klinisch verloop van mycoplasma-infecties kan aanzienlijk verschillen tussen
varkensbedrijven afhankelijk van managementstrategieën en omgevingscondities. Ook de
virulentie van de M. hyopneumoniae stammen kan de klinische uitkomst bepalen, waardoor
de controle van deze ziekteverwekker een moeilijke zaak is. Controlemaatregelen omvatten
het optimaliseren van de bedrijfsvoering en de leefomstandigheden van de dieren, het gebruik
van strategische medicatie en / of vaccinatie. De huidige controlemaatregelen zijn gunstig
vanuit economisch oogpunt, maar ze leiden niet tot een duurzame oplossing voor de ziekte.
Er is slechts beperkte informatie beschikbaar over het voorkomen van risicofactoren die
betrokken zijn bij vroege infecties en er zijn nog veel vragen over het effect van vaccinatie op
de verspreiding van de ziekteverwekker. De beperkte beschikbare studies richten zich
voornamelijk op experimentele condities en komen niet noodzakelijk overeen met de situatie
in het veld. Bovendien is informatie over de verschillen die er bestaan tussen het verloop van
infecties die veroorzaakt worden door laag en door hoog virulente stammen en het effect van
vaccinatie op infecties met verschillende M. hyopneumoniae stammen, zeer schaars.
Antwoorden op deze vragen zijn nodig voor een beter begrip van de pathogeniciteit van dit
micro-organisme, en om de bestrijding van enzoötische pneumonie te verbeteren.
Het algemene doel van het onderhavige onderzoek was om het belang van vroege infectie en
transmissie van M. hyopneumoniae onder veldomstandigheden te beoordelen en om de
bescherming tegen verschillende M. hyopneumoniae stammen te onderzoeken.
In de eerste studie (hoofdstuk 3.1), werd de prevalentie van M. hyopneumoniae bij 3-weken
oude varkens in verschillende Europese landen geschat en werden mogelijke risicofactoren
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SUMMARY - SAMENVATTING
geïdentificeerd. Op 52 bedrijven werden nasale swabs van gespeendebiggen genomen voor
analyse met behulp van nested PCR. Potentiële risicofactoren voor respiratoire aandoeningen
werden geanalyseerd met een multivariabel logistisch regressie model. Het gemiddelde
percentage positieve biggen was 10,7% (95% betrouwbaarheidsinterval, CI 7,4-14,2). Op
68% van de bedrijven testte tenminste één varken positief. Op 32% van de bedrijven bleken
meer dan 10% van de biggen positief te testen. Bedrijven met zeugen gevaccineerd tegen
swine influenza virus (SIV) hadden een significant hoger risico om biggen te hebben die
positief waren voor M. hyopneumoniae (OR 3,12, 95% CI 1.43 tot 6.83). Het hogere risico in
geval van SIV vaccinatie is moeilijk te verklaren, maar het kan te wijten zijn aan het feit dat
de varkens op bedrijven met respiratoire symptomen meer kans hebben om te worden
gevaccineerd tegen SIV waarbij de mogelijke invloed van andere respiratoire pathogenen
zoals M. hyopneumoniae over het hoofd gezien wordt. Onze resultaten bevestigen dat infectie
met M. hyopneumoniae algemeen voorkomt bij 3-weken oude biggen in verschillende
Europese landen.
Vaccinatie van biggen is één van de belangrijkste strategieën die momenteel wordt gebruikt
voor de bestrijding van M. hyopneumoniae infecties. Het is echter niet bekend of vaccinatie
van gespeende biggen de verspreiding van M. hyopneumoniae tegengaat onder
veldomstandigheden tijdens de batterijperiode.
In hoofdstuk 3.2 wordt een studie beschreven naar het effect van vaccinatie tegen M.
hyopneumoniae op de transmissie bij gespeende varkens onder veldomstandigheden.
Tweeënzeventig varkens werden bij het spenen willekeurig toegewezen aan gevaccineerde
(V) of niet-gevaccineerde (NV) groepen. Dieren in de V-groep werden gevaccineerd op de
leeftijd van 3 weken met een commercieel M. hyopneumoniae bacterin vaccin. Bij het spenen
en aan het einde van de opfokperiode werd bronchoalveolair spoelvocht genomen. Dat werd
onderzocht op de aanwezigheid van M. hyopneumoniae door middel van een nested PCR
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SUMMARY - SAMENVATTING
waarna de transmissieratio van de infectie (Rn) werd berekend. Het percentage positieve
varkens in de V en NV groepen bedroeg respectievelijk 14% en 36% bij het spenen en 31%
en 64% aan het einde van de opfokperiode. De Rn-waarden voor de V en NV groepen waren
respectievelijk 0,71 en 0,56 (P> 0,05). Uit de resultaten van deze studie kan geconcludeerd
worden dat vaccinatie de verspreiding van dit respiratoir pathogeen niet significant
vermindert.
Vervolgens (hoofdstuk 3.3) werd het infectiepatroon en de ontwikkeling van longlesies bij
varkens, geïnfecteerd met een laag of hoog virulente M. hyopneumoniae stam, op 4 en 8
weken (w) na infectie (PI) onderzocht. De werkzaamheid van een commercieel geïnactiveerd
bacterin vaccin tegen infectie met elk van deze M. hyopneumoniae stammen werd ook
bepaald. Negentig biggen die vrij waren van M. hyopneumoniae werden geselecteerd en 40
van hen werden willekeurig gevaccineerd tijdens hun eerste levensweek. Bij het spenen,
werden alle biggen toegewezen aan 10 verschillende groepen en gehuisvest in hokken met
HEPA filters. Op de leeftijd van 4 weken, werden de varkens intratracheaal geïnoculeerd met
ofwel een hoog virulente of laagvirulente stam van M. hyopneumoniae, ofwel met een steriel
groeimedium. De helft van alle dieren werd gedood op 4W PI, terwijl de andere helft werd
geëuthanaseerd op 8W PI. Dagelijks werd het hoesten geëvalueerd. Na autopsie werden een
score van de longlesies, immunofluorescentie (IF), bacteriologische analyse en nested PCR
uitgevoerd. Aangetoond werd dat de laag virulente stam, in tegenstelling tot de hoog virulente
stam, meer dan 4 weken PI (algemeen aanvaard als het standaard-infectie model) nodig heeft
om maximale klinische symptomen te veroorzaken. Vaccinatie verminderde significant de
klinische symptomen en de macroscopische en microscopische longlesies bij varkens besmet
met de hoog virulente stam. Dit effect was meer uitgesproken na 4 weken PI dan na 8 weken
PI. Bij de varkens die besmet waren met de laag virulente stam werd ook een beschermende
werking aangetoond, maar het effect was minder uitgesproken dan bij de hoog virulente stam.
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SUMMARY - SAMENVATTING
Tenslotte werd het effect van een infectie met laag virulente stammen van M. hyopneumoniae
(LV1 en LV2) op de latere infectie met een hoog virulente stam (HV) geëvalueerd (hoofdstuk
3.4). Vijfenvijftig, 3 weken oude biggen die vrij waren van M. hyopneumoniae werden
willekeurig toegewezen aan 6 verschillende groepen. Op de leeftijd van 4 weken (D0),
werden de groepen LV1-HV-en LV1 intratracheaal geïnoculeerd met LV1 en werden de
groepen LV2-HV en LV2 met LV2, en de groep HV met steriel groeimedium geïnoculeerd.
Vier weken later (D28) werden de varkens van deze verschillende groepen intratracheaal
geïnoculeerd met hetzij de hoog virulente stam (groepen LV1-HV, LV2-HV, HV) hetzij het
steriel groeimedium (groepen LV1 en LV2). Een negatieve controlegroep bestond uit varkens
geïnoculeerd met steriel groeimedium op D0 en D28. Alle dieren werden 28 dagen na de
tweede inenting geëuthanaseerd. Klinische symptomen werden geëvalueerd met behulp van
een dagelijkse hoestscore (RDS). Na autopsie werden macroscopische en histopathologische
longlesies gekwantificeerd en werden immunofluorescentie (IF) testen op het longweefsel en
nested PCR op het BAL-vloeistof uitgevoerd voor de detectie van M. hyopneumoniae.
Ziektesymptomen en longlesies werden niet waargenomen bij varkens van de negatieve
controle groep. In de andere groepen waren er geen of slechts zeer milde klinische
symptomen van D0 tot D28. Een significante stijging van de gemiddelde RDS waarden werd
echter waargenomen tijdens D28-D56, vooral in de groepen LV1-HV (1,48) en LV2-HV
(1,49), in groep HV (0,79), en in mindere mate in de groepen LV1 (0.50) en LV2 (0,65) (P
<0,05). De klinische symptomen tussen D28-D56, de longlesies en de intensiteit van de IF
kleuring waren meer uitgesproken in de groepen LV1-HV, LV2-HV en HV in vergelijking
met de groepen LV1 en LV2. Alle varkens, met uitzondering van die uit de negatieve
controlegroep, waren positief met IF testen en PCR op D56. Met deze studie werd aangetoond
dat de varkens die ingeënt waren met een laag virulente stam van M. hyopneumoniae niet
beschermd zijn tegen een volgende infectie met een hoog virulente stam, 4 weken later en
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SUMMARY - SAMENVATTING
zelfs ernstigere ziekte symptomen ontwikkelen. Dit wijst erop dat opeenvolgende infecties
met verschillende M. hyopneumoniae stammen kan leiden tot ernstige klinische ziekte op een
varkensbedrijf.
Uit het onderzoek beschreven in dit proefschrift, kan worden geconcludeerd dat M.
hyopneumoniae infecties op jonge leeftijd beginnen en wijdverspreid zijn in de Europese
varkensstapel. Vele vragen blijven nog onbeantwoord en nog veel meer gedetailleerde studies
zijn nodig om de mogelijke risicofactoren verbonden aan deze infecties te beoordelen.
Er werd geen significant verschil in transmissie van M. hyopneumoniae waargenomen tussen
biggen die gevaccineerd waren tegen M. hyopneumoniae en biggen die niet gevaccineerd
waren. Dit betekent dat vaccinatie alleen niet voldoende is om een significante daling van M.
hyopneumoniae infecties te bekomen en dat aanvullende controlemaatregelen nodig zijn om
dit pathogene agens uit de varkensstapel te elimineren. Door middel van toekomstige studies
moet nauwkeuriger het aantal M. hyopneumoniae bacteriën in de longen gekwantificeerd
worden en moet onderzocht worden of bestrijdingsmaatregelen in staat zijn om een
significante daling van de infectie in de longen te bekomen.
Het infectiepatroon en verloop zijn verschillend na experimentele infectie met een hoog of
laag virulente stam van M. hyopneumoniae, en ook de effectiviteit van vaccinatie lijkt te
worden bepaald door de aard van de stam. Een beter begrip van het infectiepatroon en de
interacties tussen hoog en laag virulente stammen is nodig. Varkens die opeenvolgend
geïnfecteerd worden met verschillende stammen ontwikkelen ernstiger klinische symptomen
en lesies. Het is echter niet bekend of gelijktijdige infectie met twee verschillende stammen
zal leiden tot een ernstiger vorm van de ziekte en of beide stammen zich in hetzelfde tempo in
het varken zullen vermenigvuldigen en/of één stam de andere zal domineren. Dit zijn
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SUMMARY - SAMENVATTING
belangrijke vragen aangezien ze kunnen helpen om het klinisch verloop van de infectie te
verklaren en om de huidige controlemaatregelen tegen enzoötische pneumonie te verbeteren.
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ACKNOLEDGEMENTS
212
ACKNOLEDGEMENTS
ACKNOLEDGEMENTS
The work here delivered would not be possible without proper framework, support and input
from a number of people. Here I want to thank everyone who helped me in one way or
another to make this thesis a reality.
First of all, I would like to thank my supervisors. Foremost, Prof. Dominiek Maes, for his
continuous support, diligence, encouragement and patience. Your time, deep knowledge,
insightful comments and ability to guide me throughout the whole doctorate period of three
years are infinitely appreciated. I would also like to show my gratitude to Prof. Haesebrouck
and Prof. Pasmans, for the discussion meetings on Mycoplasma, their guidance and helpful
suggestions in every project, as well as their revisions in every new paper. I have benefited
immensely from the extensive knowledge, experience and criticism. Thanks a lot.
I would like to acknowledge the support from IWT for the financial support of the entire
project (project number 050642). Also thanks to all the companies that made each of the
projects possible, especially Pfizer and Fort Dodge Animal Health for helping financing some
studies. Also thanks to UGent and the doctoral schools for offering different interesting
training programs.
I am also indebted to many of my colleagues, whose support has been precious. Tom, you
have made available your help in a number of ways, especially at the beginning of my PhD.
The introduction to the whole topic, to new techniques, planning and methodology of new
trials were crucial to get started and to continue later on by myself. Femke you had also
contributed substantially to my initiation in this topic, your help and guidance are equally
appreciated.
Katleen, I would like to thank you for the productive collaboration and partnership. It was
nice to have you in the same team as a discussion partner, as well as an expert in the
molecular part- my weak side . I definitely owe you one for the translation of the summary213
ACKNOLEDGEMENTS
infinite thanks for that! I wish you all the luck and success for the defense of your doctorate
next year. Keep in touch.
Dries, a big thank goes to you as well- for the collaboration, original ideas, long talks,
explanations and partnership in the myco-business. It has been nice to have shared your
knowledge, experience and friendship. You are a big fish, so find the right pond and make
sure to end up swimming in the ocean. Keep it up.
Hanne, none of the protocols written on paper would have ever seen daylight, nor any results
in practice, if it was not because of you being at the engine of the lab work. My most sincere
gratitude for your dedication, availability, assistance, patience, constancy and ever young
spirit during all this time. I honestly could not have had a better person to help me out in the
lab!
The support and encouragement of my colleagues and head of department, Prof. Aart de
Kruif, has also been indispensable; especially of those in my office and my immediate
vicinity. Ellen, Emily and Caroline, I would particularly like to thank you for the
comprehension, talks, jokes and laughs, for standing my good and bad moods, listening to my
extraterrestrial ideas, and for your prompt response to all the urgent requests. It has been a
real pleasure to have had you in the same office and to have worked with all of you. Please
come and say hello, wherever I am in the world .
I would definitely like to acknowledge the support of Alfonso. You got your hands dirty and
nearly broke your back in pretty much all of my projects. Thanks for your good sense of
humor and positive approach. The hard work would have been far from performed without
your disposal and the mega input you always provided. Aunque la formalidad acentúa
demasiado la distinción entre autor y partícipe, tu nombre merece un lugar después del mío en
la autoría de este trabajo . Un millón de gracias.
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ACKNOLEDGEMENTS
Igualmente Rubén, agradezco muchísimo la buena disposición y la mano que me prestaste en
la parte clínica de mis experimentos. Andres, creo que aún no te he visto en los
acknowledgements de una tesis , pero se te hecha de menos por las ideas desmesuradas, tu
enfoque de naturopatólogo, la ‗chimba‘ y buen sentido del humor. Buena suerte con el PhD.
I also appreciate the good collaboration of all those who helped me in both, the Department of
Reproduction, Obstetrics and Herd Health, as well as in the Department of Pathology,
Bacteriology and Avian diseases.
Thanks to all those who directly or indirectly were involved and contributed with their
knowledge and skills to this work, Prof. Richard Ducatelle, Prof. Koen Chiers, Prof. Luc
Duchateau, Christian Puttevils, Sarah Loomans and many others.
My parents and friends cannot escape this list, for the long hours of listening and for having
been a torch in my life, a constant well of wisdom and emotional/moral support. Z serca Wam
dziękuję za dane mi życie i wychowanie, za pomoc, opiekę i zrozumienie,
za ciepło, miłość i oparcie w podejmowaniu ważnych decyzji. This thesis would certainly not
have existed without them. And of course, most certainly Raki- you got the golden globe here,
for the direct daily impact of the load in this process. Senza dubbio, sarai piu contento e
sollevato di me dopo la fine di questo dottorato. You have really been a main pillar, always
present, a very important source of positivism, patience, love, warmth and encouragement
during the tough moments. Non finiro‘ mai di ringraziarti, di cuore. Sei un grande!
Lastly, I offer my regards and blessings to all of those whose names I may have not recalled,
but who have supported me in any respect during the completion of this thesis.
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CURRICULUM VITAE
216
CURRICULUM VITAE
CURRICULUM VITAE
Iris Villarreal was born on March 21st 1983, in Warsaw, Poland. Her upbringing had an
international background and she spent many years of her childhood in Panama. She finalized
her secondary studies in 2001 with an International Baccalaureate diploma from the United
World College of the Atlantic in the United Kingdom. Her veterinary studies were undertaken
partly at Warsaw University of Life Sciences, Poland, and partly at Università di Bologna,
Italy- receiving her DVM diploma in July 2007. During her years of study, she carried out
clinical practice on small and exotic animals in countries like Singapore, UK and Panama, as
well as a research project on the binding specificity of immunoglobulins and receptors
encoded by cytomegalovirus at the University of Cambridge.
Her interest in research led her to start a PhD program, as part of a IWT research project, in
January 2008 at the Faculty of Veterinary Medicine in Ghent University. The title of the
project was: ―Study of the diversity of Mycoplasma hyopneumoniae as basis for a better
prevention of enzootic pneumonia in pigs‖. The research took place at the Department of
Reproduction, Obstetrics and Herd Health and the Department of Pathology, Bacteriology and
Poultry diseases.
Iris is first author and co-author of several articles published in international peer reviewed
journals. Her experimental work has been presented in different European and international
congresses.
217
BIBLIOGRAPHY
218
BIBLIOGRAPHY
BIBLIOGRAPHY
ARTICLES
Villarreal, I., Maes, D., Meyns, T., Gebruers, F., Calus, D., Pasmans, F., Haesebrouck, F.,
2009. Infection with a low virulent Mycoplasma hyopneumoniae isolate does notprotect
piglets against subsequent infection with a highly virulent M. hyopneumoniae isolate.
Vaccine, 27, 1875-1879.
Villarreal, I., Vranckx, K, Duchateau, L., Pasmans, F., Haesebrouck, F., Jensen, J., Nanjiani,
I.A., Maes, D., 2010. Early Mycoplasma hyopneumoniae infections in suckling pigs in herds
with respiratory problems in Europe: detection rate and risk factors. Veterinarni Medicina, 55,
318–324.
Villarreal, I., Meyns, T., Dewulf, J., Vranckx, K., Calus, D., Pasmans, F., Haesebrouck, F.,
Maes, D., 2010. Effect of vaccination against Mycoplasma hyopneumoniae on the
transmission of M. hyopneumoniae under field conditions. The Veterinary Journal, accepted,
article in press.
Villarreal, I., Maes, D, Vranckx, K., Calus, D., Pasmans, F., Haesebrouck, F., 2010. Effect of
vaccination of pigs against experimental infection with highly and low virulent Mycoplasma
hyopneumoniae strains. Vaccine, article submitted.
Calus D., Maes D., Villarreal I., Vranckx K., Pasmans F., Haesebrouck F., 2010.
Determination of the growth characteristics of Mycoplasma hyopneumoniae field isolates of
different virulence by ATP luminometry and the color changing units assay. Journal of
Microbiological Methods, accepted, article in press.
ORAL PRESENTATIONS
Villarreal, I., Maes, D., Meyns, T., Gebruers, F., Calus, D., Pasmans, F., Haesebrouck, F.
Effect of a previous infection with a low virulent Mycoplasma hyopneumoniae strain on the
course of an infection with a highly virulent M. hyopneumoniae strain. In: Proc. 20th
Conference of the International Pig Veterinary Society (IPVS), June 22-26, 2008, SouthAfrica, pp. 108.
Villarreal I, Vranckx K., Duchateau Luc, Pasmans F, Haesebrouck F, Jensen J, Nanjiani I,
Maes D. Prevalence and risk factors for early Mycoplasma hyopneumoniae infections in
suckling piglets in different EU countries. Conference of the International Pig Veterinary
Society (IPVS), July 18-21, 2010,Vancouver, Canada, pp. 137
Villarreal I., Maes D., Vranckx K., Pasmans F., Haesebrouck, F. Effect of vaccination against
experimental infection with highly and low virulent Mycoplasma hyopneumoniae strains.
Conference of the International Pig Veterinary Society (IPVS), July 18-21, 2010,Vancouver,
Canada, pp. 136
POSTERS AND PUBLICATIONS IN PROCEEDINGS OF NATIONAL AND
INTERNATIONAL CONGRESSES
Villarreal I., Gebruers F., Meyns T., Calus D., Pasmans F., Haesebrouck F., Maes D. Effect of
a previous infection with a low virulent Mycoplasma hyopneumoniae isolate on the course of
219
BIBLIOGRAPHY
an infection with a highly virulent M. hyopneumoniae isolate. In: Proc. 17th Conference of the
International Organisation of Mycoplasmology (IOM), July 6-11, 2008, Tianjin, China, pp.
91.
Villarreal, I., Jensen J.C.E., Nanjiani I.A., Maes D. Prevalenza dell‘infezione da Mycoplasma
hyopneumoniae in suini di tre settimane di età in diversi paesi europei. Atti della Societa
Italiana di Patologia ed allevamento dei suini. Meeting annuale, March 10-13, 2009. Modena,
Italy, pp. 527.
Villarreal I., Maes D., Vranckx K., Pasmans F., Haesebrouck, F. Effect of vaccination against
Mycoplasma hyopneumoniae on its transmission under field conditions. Conference of the
International Pig Veterinary Society (IPVS), July 18-21, 2010, Vancouver, Canada.
Gebruers, F., Calus, D., Maes, D., Villarreal, I. et al. A set of four nested PCRs to detect
different strains of Mycoplasma hyopneumoniae in biological samples. International
Organisation of Mycoplasmology (IOM), July 6-11, 2008, Tianjin, China.
Vranckx K., Maes D., Villareal I., Calus D., Pasmans F., Haesebrouck F. A multiple-locus
variable-number tandem-repeat analysis for the study of the diversity of Mycoplasma
hyopneumoniae. In: Proc. Mycoplasmology: A review of developments over the last decade,
June 10-12, 2009, Gran Canaria, Spain, pp. 63.
Vranckx K., Maes D., Villareal I., Calus D., Pasmans F., Haesebrouck F. A multiple-locus
variable-number tandem-repeat analysis for the study of the diversity of Mycoplasma
hyopneumoniae. In: Proc. International Pig Veterinary Society (IPVS) Belgian branch,
November 27, 2009, Merelbeke, Belgium, poster 19.
220
The difference between involvement and commitment is like with
"Ham & Eggs: a day‘s work for a chicken, but a lifetime commitment for a pig."
221