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FACULTY OF VETERINARY MEDICINE<br />
<strong>Department</strong> <strong>of</strong> <strong>Reproduction</strong>, <strong>Obstetrics</strong> <strong>and</strong> <strong>Herd</strong> <strong>Health</strong><br />
<strong>Department</strong> <strong>of</strong> Pathology, Bacteriology <strong>and</strong> Poultry Diseases<br />
Virulence <strong>and</strong> antimicrobial susceptibility <strong>of</strong> Mycoplasma<br />
hyopneumoniae isolates from pigs<br />
Jo VICCA<br />
Thesis to obtain the academic degree <strong>of</strong> Doctor <strong>of</strong> Veterinary Science (PhD)<br />
Faculty <strong>of</strong> Veterinary Medicine, Ghent University<br />
2005<br />
Promotoren: Pr<strong>of</strong>. Dr. F. Haesebrouck, Pr<strong>of</strong>. Dr. D. Maes<br />
Co-promotor: Pr<strong>of</strong>. Dr. Dr.h.c. A. de Kruif
FRONT COVER: Male pig kept for several months as blood donor for in vitro studies with M. hyopneumoniae. Unfortunately,<br />
the results did not satisfy <strong>and</strong> are not included in this PhD thesis. To my opinion, the pigs do deserve a place in this thesis <strong>and</strong><br />
therefore reached the cover.<br />
BACK COVER: - Me together with my blood donating pigs<br />
- Scanning electron microscopic photograph <strong>of</strong> a M. hyopneumoniae colony grown on agar<br />
- Cryosection <strong>of</strong> M. hyopneumoniae infected lung tissue labeled with anti-M. hyopneumoniae<br />
antibodies <strong>and</strong> showing positive immun<strong>of</strong>luorescence<br />
- Section <strong>of</strong> paraffin embedded M. hyopneumoniae infected lung tissue, stained with hematoxylineosin,<br />
showing the typical peribronchiolar hyperplasia compressing the involved airway<br />
PRINTING: Plot-it Merelbeke, 2005<br />
ISBN: 90-5864-086-8<br />
EAN: 9789058640864
Wisdom is not a question <strong>of</strong> learning facts with the mind; it can only be<br />
acquired through perfection <strong>of</strong> living.<br />
N. Sri Ram
CONTENTS<br />
List <strong>of</strong> abbreviations 1<br />
1. INTRODUCTION 3<br />
1.1 Re<strong>view</strong> <strong>of</strong> the literature 5<br />
1.1.1 Pathogenesis <strong>of</strong> enzootic pneumonia 11<br />
1.1.2 Control <strong>and</strong> treatment <strong>of</strong> enzootic pneumonia in pigs <strong>and</strong><br />
in vitro susceptibility <strong>of</strong> M. hyopneumoniae 27<br />
1.2 Aims 59<br />
2. EXPERIMENTAL STUDIES 63<br />
2.1 Virulence <strong>of</strong> Mycoplasma hyopneumoniae isolates 65<br />
2.1.1 Patterns <strong>of</strong> Mycoplasma hyopneumoniae infections in Belgian<br />
farrow-to-finish pig herds with diverging disease course 67<br />
2.1.2 Evaluation <strong>of</strong> virulence <strong>of</strong> Mycoplasma hyopneumoniae field<br />
isolates 85<br />
2.2 Susceptibility <strong>of</strong> Mycoplasma hyopneumoniae to antimicrobial agents 107<br />
2.2.1 In vitro susceptibilities <strong>of</strong> Mycoplasma hyopneumoniae field<br />
isolates 109<br />
2.2.2 Characterization <strong>of</strong> in vivo acquired resistance <strong>of</strong> Mycoplasma<br />
hyopneumoniae to macrolides <strong>and</strong> lincosamides 125<br />
2.2.3 Mechanism <strong>of</strong> resistance against the fluoroquinoles flumequine<br />
<strong>and</strong> enr<strong>of</strong>loxacin in Mycoplasma hyopneumoniae field isolates 139<br />
2.2.4 The efficacy <strong>of</strong> tylosin premix for the treatment <strong>and</strong> control <strong>of</strong><br />
Mycoplasma hyopneumoniae infections 157<br />
3 GENERAL DISCUSSION 175<br />
4 SUMMARY 187<br />
5 SAMENVATTING 195<br />
Dankwoord 203<br />
Curriculum vitae 209<br />
Publications 213
LIST OF ABBREVIATIONS<br />
A: Adenine<br />
ABC:<br />
ATP-Binding Cassette<br />
ADG:<br />
Average Daily weight Gain<br />
AIAO:<br />
All-in/All-out<br />
AMK:<br />
Amoxycillin<br />
ANOVA:<br />
ANalysis Of VAriance<br />
APC:<br />
Antigen-Presenting Cell<br />
APR:<br />
Apramycin<br />
ATCC:<br />
American Type Culture Collection<br />
ATP:<br />
Adenosine triphosphate<br />
BALT:<br />
Bronchus-Associated Lymphoid Tissue<br />
bp:<br />
base pair<br />
BW:<br />
Body Weight<br />
C: Cytosine<br />
CD:<br />
Cluster <strong>of</strong> Differentiation<br />
CCU:<br />
Colour Changing Units<br />
CEF:<br />
Cefquinome<br />
CHLO:<br />
Chloortetracycline<br />
CIPRO:<br />
Cipr<strong>of</strong>loxacin<br />
CLIN:<br />
Clindamycin<br />
COL:<br />
Colistin<br />
DANO:<br />
Dan<strong>of</strong>loxacin<br />
DNA:<br />
DeoxyriboNucleic Acid<br />
DOX:<br />
Doxycycline<br />
DWG:<br />
Daily Weight Gain<br />
E. coli: Escherichia coli<br />
ELISA:<br />
Enzyme-Linked ImmunoSorbent Assay<br />
ENRO:<br />
Enr<strong>of</strong>loxacin<br />
FCR:<br />
Feed Conversion Ratio<br />
FFN:<br />
Florfenicol<br />
FLUM:<br />
Flumequin<br />
G: Guanine<br />
GEN:<br />
Gentamicin<br />
G-F:<br />
Growth-Finishing<br />
HEPA:<br />
High Efficiency Particulate Air<br />
IF:<br />
Immun<strong>of</strong>luorescence<br />
Ig:<br />
Immunoglobulin<br />
IL:<br />
Interleukin<br />
IL-R:<br />
Interleukin-Receptor<br />
IM:<br />
Intramuscular<br />
IRPCM:<br />
International Research Programme on Comparative Mycoplasmology<br />
K+:<br />
kalium ion<br />
kDa:<br />
kilo Dalton<br />
LIN:<br />
Lincomycin<br />
MALP:<br />
Macrophage Activating LipoProtein<br />
Mbp:<br />
Million base pairs<br />
MCP:<br />
Monocyte chemoattractant protein<br />
MEW:<br />
Medicated Early Weaning<br />
Mh:<br />
Mycoplasma hyopneumoniae<br />
MHC:<br />
Major Histocompatibility Complex<br />
M. hyopneumoniae: Mycoplasma hyopneumoniae<br />
MIC:<br />
Minimum Inhibitory Concentration<br />
MIP:<br />
Macrophage Inflammatory Protein<br />
MLS:<br />
Macrolides, Lincosamides <strong>and</strong> Streptogramins<br />
MPC:<br />
Mutant Prevention Concentration<br />
mRNA:<br />
messenger RiboNucleic Acid<br />
- 1 -
N: Nursery<br />
NCCLS:<br />
National Committee for Clinical Laboratory St<strong>and</strong>ards<br />
NK cells:<br />
Natural Killer cells<br />
NOR:<br />
Norfloxacin<br />
nPCR:<br />
nested Polymerase Chain Reaction<br />
OD:<br />
Optical Density<br />
OFLO:<br />
Ofloxacin<br />
OXY:<br />
Oxytetracycline<br />
PBS:<br />
Phosphate Buffered Saline<br />
PCR:<br />
Polymerase Chain Reaction<br />
PEN:<br />
Penicillin<br />
PFGE<br />
Pulsed Field Gel Electrophoresis<br />
ppm:<br />
parts per million<br />
PRRSV:<br />
Porcine Reproductive <strong>and</strong> Respiratory Syndrome Virus<br />
RAPD:<br />
R<strong>and</strong>omly Amplified Polymorphic DNA<br />
rDNA:<br />
ribosomal DeoxyriboNucleic Acid<br />
RDS:<br />
Respiratory Disease Score<br />
R n :<br />
Adjusted reproduction ratio<br />
RNA:<br />
RiboNucleic Acid<br />
rpm:<br />
revolutions per minute<br />
RR:<br />
Repeat Region<br />
rRNA:<br />
ribosomal RiboNucleic Acid<br />
QRDR:<br />
Quinolone Resistance Determining Region<br />
30S: 30 Svedberg units<br />
SD:<br />
St<strong>and</strong>ard Deviation<br />
SPIRA:<br />
Spiramycin<br />
SPT:<br />
Spectinomycin<br />
STR:<br />
Streptomycin<br />
SULF:<br />
Sulfamides<br />
T: Thymine<br />
TBE:<br />
Tris-Borate-EDTA<br />
TEM:<br />
Transmission Electron Microscopy<br />
TET:<br />
Tetracycline<br />
Th-cell:<br />
T helper-cell<br />
TIA:<br />
Tiamulin<br />
TIL:<br />
Tilmicosin<br />
TLR:<br />
Toll-Like Receptor<br />
TMP:<br />
Trimethoprim<br />
TNF:<br />
Tumor Necrosis Factor<br />
TP:<br />
Tylosin-Premix<br />
tRNA:<br />
transfer RiboNucleic Acid<br />
TYL:<br />
Tylosin<br />
VAL:<br />
Valnemulin<br />
VLA:<br />
Veterinary Laboratory Agency<br />
Vlp:<br />
Variable lipoproteins<br />
w/v:<br />
weight/volume<br />
- 2 -
- 3 -<br />
1 INTRODUCTION
1.1 REVIEW OF THE LITERATURE<br />
- 5 -
Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, a chronic<br />
respiratory disease occurring worldwide in the pig industry. Over 90% <strong>of</strong> the Belgian<br />
pig herds are endemically infected (Maes et al., 1999). The major drawbacks <strong>of</strong><br />
enzootic pneumonia are the economic losses due to decreased daily weight gain,<br />
increased feed conversion ratio, number <strong>of</strong> days to reach slaughterweight <strong>and</strong><br />
medication costs.<br />
Outbreaks <strong>of</strong> enzootic pneumonia are mainly experienced in the growing <strong>and</strong><br />
finishing units. The incubation period <strong>of</strong> the disease is 10-16 days under experimental<br />
conditions, but this period can vary greatly under natural conditions. There is no agerelated<br />
susceptibility <strong>of</strong> pigs to infection with M. hyopneumoniae, piglets in the<br />
farrowing unit as well as sows can become infected after experimental inoculation or<br />
during a natural outbreak <strong>of</strong> enzootic pneumonia (Goodwin et al., 1969; Piffer <strong>and</strong><br />
Ross, 1984; Kobisch et al., 1993; Wallgren et al., 1998).<br />
Macroscopic lesions are mainly found bilaterally in the apical, cardiac,<br />
intermediate <strong>and</strong> the anterior parts <strong>of</strong> the diaphragmatic lobes. They appear at 7-10<br />
days after experimental infection <strong>and</strong> reach their maximum extension after 4 weeks.<br />
The lesions are well demarcated from the normal tissue <strong>and</strong> consist <strong>of</strong> purple to gray<br />
areas <strong>of</strong> consolidation. After incision <strong>of</strong> the affected tissue, the consistency is meaty<br />
<strong>and</strong> there is a catarrhal exudate in the airways. The bronchial <strong>and</strong> mediastinal lymph<br />
nodes are <strong>of</strong>ten enlarged. Lesions become deeper red <strong>and</strong> even more demarcated<br />
when the disease evolves to the chronic stage. Less exudate is present in the airways.<br />
Recovering lesions consist <strong>of</strong> fissures <strong>of</strong> collapsed alveoli (interlobular scar<br />
retractions). Macroscopic lesions <strong>of</strong> a pure M. hyopneumoniae infection are usually<br />
resolved after 12-14 weeks but M. hyopneumoniae can still be detected (Blanchard et<br />
al., 1992; Sørensen et al., 1997). Despite the resolving <strong>of</strong> the lung lesions, the<br />
organism can be demonstrated in the lungs for at least 185 days using nested PCR on<br />
bronchial swabs (Fano et al., 2004).<br />
Because M. hyopneumoniae infections disturb the mucosal clearance, form<br />
characteristic lesions <strong>and</strong> modulate the immune system, secondary infections with<br />
Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica,<br />
Haemophilus parasuis <strong>and</strong> Streptococcus suis are common (Yagihashi et al., 1984;<br />
Ciprian et al., 1988; Asai et al., 1996; Sørensen et al., 1997).<br />
- 7 -
In the present re<strong>view</strong>, literature dealing with the pathogenesis <strong>of</strong> enzootic<br />
pneumonia will be summarized. Thereafter an over<strong>view</strong> <strong>of</strong> antimicrobial treatment<br />
<strong>and</strong> control <strong>of</strong> the disease will be presented.<br />
- 8 -
REFERENCES<br />
1. Asai, T., Okada, M., Yokomizo, Y., Sato, S. & Mori, Y. (1996). Suppressive effect <strong>of</strong><br />
bronchoalveolar lavage fluid from pigs infected with Mycoplasma hyopneumoniae on<br />
chemiluminescence <strong>of</strong> porcine peripheral neutrophils. Veterinary Immunology <strong>and</strong><br />
Immunopathology 51, 325-331.<br />
2. Blanchard, B., Vena, M.M., Cavalier, A., Le Lannic, J., Gouranton, J. & Kobisch, M.<br />
(1992). Electron microscopic observation <strong>of</strong> the respiratory tract <strong>of</strong> SPF piglets inoculated with<br />
Mycoplasma hyopneumoniae. Veterinary Microbiology 30, 329-341.<br />
3. Ciprian, A., Pijoan, C., Cruz, T., Camacho, J., Tortora, J., Colmenares, G., Lopez-Revilla,<br />
R. & De La Garza, M. (1988). Mycoplasma hyopneumoniae increases the susceptibility <strong>of</strong> pigs<br />
to experimental Pasteurella multocida pneumoniae. Canadian Journal <strong>of</strong> Veterinary Research<br />
52, 434-438.<br />
4. Fano, E., Pijoan, C. & Dee, S. (2004). Assessing the duration <strong>of</strong> Mycoplasma hyopneumoniae<br />
infection in gilts. Proceedings <strong>of</strong> the International Pig Veterinary Society, 18 th Congress, June<br />
27- Juli 1, Hamburg / Germany.<br />
5. Goodwin, R., Hodgson, R., Wittlestone, P. & Woodhams, R. (1969). Immunity in<br />
experimentally induced enzootic pneumonia <strong>of</strong> pigs. Journal <strong>of</strong> Hygiene (London) 67, 193-208.<br />
6. Kobisch, M., Blanchard, B. & Le Poitier, M.F. (1993). Mycoplasma hyopneumoniae<br />
infections in pigs: duration <strong>of</strong> the disease <strong>and</strong> resistance to reinfection. Veterinary Research 24,<br />
67-77.<br />
7. Maes, D. (1999). Respiratory disease in slaughter pigs: epidemiological aspects <strong>and</strong> effect <strong>of</strong><br />
vaccination against Mycoplasma hyopneumoniae. PhD Thesis / Ghent University / Belgium.<br />
8. Piffer, I. & Ross, R. (1984). Effect <strong>of</strong> age on susceptibility <strong>of</strong> pigs to Mycoplasma<br />
hyopneumoniae pneumonia. American Journal <strong>of</strong> Veterinary Research 45, 478-481.<br />
9. Sørensen, V., Ahrens, P., Barfod, K., Feenstra, A.A., Feld, N.C., Friis, N.F., Bille-Hansen,<br />
V., Jensen, N.E. & Pedersen, M.W. (1997). Mycoplasma hyopneumoniae infection in pigs:<br />
duration <strong>of</strong> the disease <strong>and</strong> evaluation <strong>of</strong> four diagnostic assays. Veterinary Microbiology 54,<br />
23-34.<br />
10. Wallgren, O., Bolske, G., Gustafsson, S., Mattsson, S., Fossum, C. (1998). Humoral immune<br />
responses to Mycoplasma hyopneumoniae in sows <strong>and</strong> <strong>of</strong>fspring following an outbreak <strong>of</strong><br />
mycoplasmosis. Veterinary Microbiology 60, 193-205.<br />
11. Yagihashi, T., Nunoya, T., Mitui, T. & Tajima, M. (1984). Effect <strong>of</strong> Mycoplasma<br />
hyopneumoniae on the development <strong>of</strong> Haemophilus pleuropneumoniae pneumonia in pigs. The<br />
Japanese Journal <strong>of</strong> Veterinary Science 46, 705-713.<br />
- 9 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
- 11 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
Mycoplasmas are the smallest <strong>and</strong> simplest self-replicating organisms <strong>and</strong> are<br />
distinguished phenotypically from other bacteria by their minute size <strong>and</strong> lack <strong>of</strong> a<br />
cell wall. They are characterized by their small genome size (0.58-2.2 Mbp) <strong>and</strong> a low<br />
G+C content (23-40 mol% <strong>of</strong> the genome). During their evolution, Mycoplasma spp.<br />
have lost all the genes involved in amino acid <strong>and</strong> fatty acid biosynthesis <strong>and</strong> thus<br />
require the full spectrum <strong>of</strong> the essential amino acids <strong>and</strong> fatty acids from the host or<br />
from the artificial culture medium. Also genes involved in c<strong>of</strong>actor biosynthesis are<br />
lost, so that to cultivate Mycoplasma spp. in vitro, the medium has to be supplemented<br />
with essentially all the vitamins (Razin et al., 1998). Competition between<br />
mycoplasma <strong>and</strong> host for these biosynthetic precursors may lead to disruption <strong>of</strong> the<br />
host cell integrity <strong>and</strong> alter host cell function (Rottem, 2003).<br />
ADHESION OF M. HYOPNEUMONIAE TO THE HOST CELLS<br />
Adhesion <strong>of</strong> Mollicutes to host cells is a prerequisite for colonization <strong>and</strong> for<br />
infection. The loss <strong>of</strong> adhesion capacity by several passages in vitro results in loss <strong>of</strong><br />
infectivity (Mebus <strong>and</strong> Underdahl, 1977; Thomas et al., 2003). M. hyopneumoniae<br />
adheres to the apex <strong>of</strong> cilia <strong>of</strong> tracheal, bronchial <strong>and</strong> bronchiolar epithelial cells but<br />
does not penetrate into the lung parenchyma nor invade cells (Blanchard et al., 1992;<br />
Kwon <strong>and</strong> Chae, 1999). Very few mycoplasmas are present in the small bronchioli<br />
<strong>and</strong> alveoli since ciliated epithelium is absent in the lower respiratory tract<br />
(Christensen et al., 1999). M. hyopneumoniae exclusively adheres to cilia <strong>and</strong><br />
therefore uses fine fibrils (Tajima <strong>and</strong> Yagihashi, 1982; Blanchard et al., 1992;<br />
Sarradell et al., 2003) (Figure 1). Using electron microscopy, Tajima <strong>and</strong> Yagihashi<br />
(1982) <strong>and</strong> Tajima et al. (1985) observed the presence <strong>of</strong> a capsule <strong>and</strong> found an<br />
association between the thickness <strong>of</strong> this polysaccharide capsule <strong>and</strong> the pathogenicity<br />
<strong>of</strong> the organism.<br />
- 13 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
Figure 1: Adapted from Tajima <strong>and</strong> Yagihashi (1982). A TEM photograph <strong>of</strong> a crosssection<br />
<strong>of</strong> the bronchiolar epithelium from a M. hyopneumoniae inoculated pig.<br />
Numerous mycoplasmas are seen among the cilia <strong>and</strong> on the tips <strong>of</strong> microvilli <strong>of</strong> the<br />
epithelial cells. The organisms are not in contact with the epithelial surface. Bar:<br />
1.000 nm. The inset shows a mycoplasma which is elongated <strong>and</strong> constricted in the<br />
middle part <strong>of</strong> the body <strong>and</strong> appears to be in the process <strong>of</strong> binary fission. Many radial<br />
fibrils can be seen projecting outward from the cell surface. Bar: 100 nm.<br />
Still little is known about the mechanism <strong>of</strong> adherence <strong>of</strong> M. hyopneumoniae.<br />
It is clear that the process is multifactorial <strong>and</strong> that multiple genes are involved.<br />
Zhang et al. (1995) identified a ciliary adhesion molecule (P97). Monoclonal<br />
antibodies against P97 inhibited adherence <strong>and</strong> stained the fuzzy structures, as<br />
described by Tajima <strong>and</strong> Yagihashi (1982), on the surface <strong>of</strong> the micro-organism.<br />
Further analysis <strong>of</strong> the DNA sequences surrounding the P97 structural gene revealed<br />
an operon composed <strong>of</strong> two open reading frames encoding the P97 adhesion molecule<br />
<strong>and</strong> a 102 kDA protein (P120). The role <strong>of</strong> P102 is unknown. P97 includes two repeat<br />
regions, R1 <strong>and</strong> R2. (Hsu et al., 1997; Hsu <strong>and</strong> Minion, 1998a). The cilium binding<br />
site is located in R1 <strong>and</strong> it is supposed that at least seven AAKPV(E) repeats are<br />
required for functional binding (Hsu <strong>and</strong> Minion, 1998b; Minion et al. 2000).<br />
A glycoprotein <strong>of</strong> 110 kDa, consisting <strong>of</strong> one P54 <strong>and</strong> two P28 subunits is also<br />
associated with adhesion <strong>of</strong> M. hyopneumoniae organisms to the cilia (Chen et al.,<br />
1998).<br />
- 14 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
Adherence is associated with degenerative changes in the ciliated epithelial<br />
cells: reduction <strong>of</strong> the ciliary activity, loss <strong>of</strong> cilia <strong>and</strong> clumping <strong>of</strong> cilia. By attaching<br />
to the surface <strong>of</strong> host cells, mycoplasmas may interfere with membrane receptors or<br />
alter transport mechanisms <strong>of</strong> the host cell. In the case <strong>of</strong> M. hyopneumoniae, a<br />
disruption <strong>of</strong> K+ channels <strong>of</strong> ciliated bronchial epithelial cells which results in<br />
ciliostasis has been described (Debey <strong>and</strong> Ross, 1994). Recent research revealed the<br />
increase <strong>of</strong> intracellular calcium in porcine ciliated tracheal cells after adhesion <strong>of</strong><br />
pathogenic M. hyopneumoniae isolates (Park et al., 2002). This increase may serve as<br />
an intracellular signal to induce loss <strong>of</strong> cilia. Cell damage may also be caused by the<br />
mildly toxic by-products <strong>of</strong> mycoplasma metabolism, such as hydrogen peroxide <strong>and</strong><br />
superoxide radicals. In addition, cytotoxic factors have been associated with some<br />
Mycoplasma spp., including M. fermentans (Gabridge <strong>and</strong> Murphy, 1971; Gabridge<br />
<strong>and</strong> Schneider, 1975), M. hyorhinis (Darai et al., 1981) M. neurolyticum (Tully, 1981)<br />
<strong>and</strong> M. hyopneumoniae (Geary <strong>and</strong> Walczak, 1985).<br />
In the host, receptors for mycoplasmas are usually <strong>of</strong> sialoglycoconjugate<br />
nature (Razin <strong>and</strong> Jacobs, 1992; Zhang et al., 1994). The restricted attachment <strong>of</strong> M.<br />
hyopneumoniae to cilia can partially be explained by the presence <strong>of</strong> these<br />
sialoglycoconjugate-type receptors on the apical microvillar border <strong>and</strong> the cilia, <strong>and</strong><br />
the absence on the secretory cells <strong>and</strong> mucus (Loveless <strong>and</strong> Feizi, 1989).<br />
MICROSCOPIC LESIONS ASSOCIATED WITH M. HYOPNEUMONIAE INFECTION<br />
Histological lesions in the acute stage <strong>of</strong> the disease include accumulation <strong>of</strong><br />
neutrophils <strong>and</strong> macrophages in <strong>and</strong> around airways (Livingston et al., 1972). As the<br />
disease progresses (7-28 days post infection), the changes consist <strong>of</strong> infiltrates <strong>of</strong><br />
mononuclear cells (lymphocytes <strong>and</strong> macrophages) around bronchi, bronchioles <strong>and</strong><br />
small blood vessels, accumulation <strong>of</strong> inflammatory mononuclear cells forming<br />
lymphoid follicles around the airways <strong>and</strong> prominent lymphoid hyperplasia <strong>of</strong> the<br />
lymphoid tissue associated with intrapulmonary airways, known as bronchusassociated<br />
lymphoid tissue (BALT) (Huang et al., 1990) (Figure 2). In alveolar<br />
lumina <strong>and</strong> septa, lymphocytes, plasma cells <strong>and</strong> neutrophils accumulate (Rodríguez<br />
et al., 2004). This accumulation aggravates obliteration <strong>of</strong> the lumen <strong>of</strong> bronchioles<br />
<strong>and</strong> atelectasis <strong>of</strong> surrounding alveoli.<br />
- 15 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
Figure 2: Infection with M. hyopneumoniae is characterized by infiltration <strong>of</strong><br />
mononuclear cells around bronchi, bronchioles <strong>and</strong> small blood vessels forming<br />
lymphoid follicles. This accumulation aggravates obliteration <strong>of</strong> the lumen <strong>of</strong> bronchi<br />
<strong>and</strong> bronchioles (arrow) <strong>and</strong> small blood vessels. Hematoxilin-Eosin X 400.<br />
Four mechanisms may contribute to the obliteration <strong>of</strong> airways: (1)<br />
accumulation <strong>of</strong> mucus <strong>and</strong> inflammatory exudates due to loss <strong>of</strong> mucociliary<br />
function, (2) increased activity <strong>of</strong> mucus-secreting cells <strong>and</strong> altered glycoprotein<br />
production in goblet cells, (3) broncho-constriction by chemical mediators released by<br />
alveolar macrophages <strong>and</strong> inflammatory cells <strong>and</strong> (4) pressure from the aggregates <strong>of</strong><br />
lymphoid tissue (Sarradell et al., 2003).<br />
- 16 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
INTERACTION OF M. HYOPNEUMONIAE WITH THE IMMUNE SYSTEM<br />
The complex network <strong>of</strong> interactions between mycoplasmas <strong>and</strong> the host<br />
immune system involves mycoplasma-induced non-specific <strong>and</strong> specific immune<br />
reactions. Non-specific mechanisms include (1) increase <strong>of</strong> the cytotoxicity <strong>of</strong><br />
macrophages, natural killer cells <strong>and</strong> T-cells, (2) enhancement <strong>of</strong> the expression <strong>of</strong><br />
cell receptors <strong>and</strong> activating the complement cascade <strong>and</strong> (3) polyclonal activation <strong>of</strong><br />
B- <strong>and</strong> T-lymphocytes. Specific protective defense mechanisms include (1)<br />
stimulation <strong>of</strong> cell-mediated immunity, (2) opsonisation <strong>and</strong> phagocytosis <strong>of</strong><br />
organisms <strong>and</strong> (3) the production <strong>of</strong> systemic as well as local anti-mycoplasmal<br />
antibodies <strong>of</strong> different classes <strong>and</strong> subclasses. These immune reactions are important<br />
in host defense but have also been shown to play a role in the development <strong>of</strong> lesions<br />
<strong>and</strong> the exacerbation <strong>of</strong> mycoplasma induced disease (Razin et al., 1998).<br />
After colonizing the respiratory tract, M. hyopneumoniae stimulates<br />
macrophages to secrete cytokines (Asai et al., 1993, Rodríguez et al., 2004). Proinflammatory<br />
cytokines (IL-1, IL-6 <strong>and</strong> TNF-α) exert a non-specific mitogenic<br />
activity <strong>of</strong> M. hyopneumoniae on lymphocytes.<br />
Lymphocyte recruitment <strong>and</strong> activation are important in the pathogenesis <strong>of</strong><br />
mycoplasma respiratory disease. T-cells are an important component <strong>of</strong> these<br />
responses. In M. hyopneumoniae infected animals, CD4+ Th-cells represent the<br />
predominant subset responsible <strong>of</strong> the observed BALT hyperplasia (Saradell et al.,<br />
2003). CD4+ Th-cells are capable <strong>of</strong> activating macrophages, which results in<br />
efficient intracellular killing <strong>of</strong> the mycoplasmas (Caruso <strong>and</strong> Ross, 1990; Messier et<br />
al., 1990; Asai et al., 1994). Using in situ hybridization, Kwon <strong>and</strong> Chae (1999) were<br />
able to demonstrate the presence <strong>of</strong> M. hyopneumoniae DNA in interstitial <strong>and</strong><br />
alveolar macrophages. In mice, CD4+ Th-cells play a role in immune mediated<br />
pathogenesis <strong>of</strong> mycoplasmal disease since CD4+ Th-cell depleted mice develop<br />
milder pulmonary lesions than immunocompetent mice (Jones et al., 2002). Both<br />
Th1- <strong>and</strong> Th2-type cytokine responses develop in mycoplasma respiratory disease <strong>and</strong><br />
these responses are associated with disease severity. Infection results in a shift from<br />
the resident Th2 population to a mixed Th1/Th2 response. CD8+ T-<br />
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1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
cells are also observed in M. hyopneumoniae infected lungs (Sarradell et al., 2003),<br />
but less numerous compared to the CD4+ subpopulation. These cells were shown to<br />
dampen mycoplasma-associated proinflammatory responses during in vivo<br />
experimental studies in mice (Jones et al., 2002). The recruitment <strong>of</strong> B-lymphocytes<br />
results in locally secreted IgA en IgG (Messier et al., 1990). IgA prevents the<br />
adhesion <strong>of</strong> mycoplasmas to the ciliated epithelium, <strong>and</strong> IgG participates in<br />
opsonization <strong>and</strong> phagocytosis by alveolar macrophages (Sheldrake, 1990; Sheldrake<br />
et al., 1993; Walker et al., 1996). Local humoral immune response can be protective<br />
before exposure to mycoplasmas (Okada et al., 2000; Fagan et al., 2001) or can<br />
prevent the spread <strong>of</strong> the organism to other tissues. Locally secreted antibodies appear<br />
to play a limited role in recovery from mycoplasma infection since mycoplasmas can<br />
survive despite vigorous local antibody responses in the host (Simecka et al., 1993;<br />
Djordjevic et al., 1997). For several respiratory mycoplasmal diseases, locally<br />
produced antibodies are more important than circulating antibodies (Hodge <strong>and</strong><br />
Simecka, 2002).<br />
Recently, research aimed to further clarify the trigger for inflammatory cell<br />
influx after M. hyopneumoniae infection by studying the role <strong>of</strong> pro-inflammatory<br />
cytokines, which are believed to play a prominent role in the disease symptoms <strong>of</strong><br />
enzootic pneumonia. Lipoproteins are probably the principle components <strong>of</strong> intact<br />
mycoplasma organisms that activate macrophages <strong>and</strong> they may play an important<br />
role in the inflammatory process (Chambaud et al., 1999). After interaction with<br />
ciliated epithelial cells, M. hyopneumoniae causes an increase <strong>of</strong> the proinflammatory<br />
cytokines IL-1, IL-6 <strong>and</strong> TNF-α (Asai et al., 1993; 1994;<br />
Thanawongnuwech et al., 2001; Rodríguez et al., 2004). The main biological actions<br />
<strong>of</strong> the pro-inflammatory cytokines are summarized in Table 1.<br />
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1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
Table 1: Biological actions <strong>of</strong> IL-1, IL-6 <strong>and</strong> TNF-α (adapted from Henderson et al.,<br />
1996).<br />
Biological action<br />
Action displayed by:<br />
IL-1 IL-6 TNF-α<br />
Actions involved in lesion development after infection with M. hyopneumoniae<br />
Activation <strong>of</strong> T- <strong>and</strong> B-lymphocytes + + +<br />
Stimulation <strong>of</strong> immunoglobulin synthesis - + -<br />
Upregulation <strong>of</strong> cytocidal activity <strong>of</strong> macrophages <strong>and</strong> + - +<br />
NK cells<br />
Increased expression <strong>of</strong> MHC antigen on APC + - +<br />
Stimulation <strong>of</strong> cyclooxygenase II induction + - +<br />
Activation <strong>of</strong> endothelial cells + - +<br />
Induction <strong>of</strong> endothelial adhesion molecules + - +<br />
Induction <strong>of</strong> IL-1, TNF-α <strong>and</strong> IL-8 + - +<br />
Induction <strong>of</strong> IL-6 + - +<br />
Induction <strong>of</strong> IL-2 <strong>and</strong> IL-2R - + -<br />
Endogenous pyrogen + + +<br />
Additional actions with no direct impact on lesion development after infection<br />
with M. hyopneumoniae<br />
Induction <strong>of</strong> acute-phase proteins + + +<br />
Stimulation <strong>of</strong> fibroblast proliferation + + +<br />
Stimulation <strong>of</strong> hematopoiesis + + +<br />
Stimulation <strong>of</strong> cartilage breakdown + - +<br />
Stimulation <strong>of</strong> murine bone breakdown + + +<br />
Induction <strong>of</strong> septic-shock like syndrome + - +<br />
Induction <strong>of</strong> hyperalgesia + - +<br />
A macrophage activating lipoprotein 2 kDa (MALP-2), characterized in<br />
Mycoplasma fermentans (Mühlradt et al., 1997), was found to be a potent inducer in<br />
vitro <strong>and</strong> in vivo <strong>of</strong> the chemokines macrophage inflammatory protein 1α (MIP-1α),<br />
monocyte chemoattractant protein 1 (MCP-1) <strong>and</strong> MIP-2 (Deiters <strong>and</strong> Mühlradt,<br />
1999, Lührmann et al., 2002). This diacylated lipoprotein, integrated in the membrane<br />
<strong>of</strong> mycoplasmas, is recognized by Toll-like receptors (TLR)2 / TLR6. This<br />
recognition leads to a cascade reaction resulting in apoptotic cell death, complement<br />
activation or cytokine production (Akira <strong>and</strong> Hemmi, 2003; Into et al., 2004). TLR<br />
families expressed in the antigen-presenting cells (APCs) are pattern recognition<br />
molecules that recognize foreign antigens during innate immune responses <strong>of</strong> the host<br />
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1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
animals. MALP-2 like structures have been found in other mycoplasmas as well,<br />
including M. arthritidis (Cole, 1991), M. hyorhinis (Mühlrath et al., 1998) <strong>and</strong> M.<br />
salivarum (Shibata et al., 2000; Okusawa et al., 2004). Several lipoproteins in M.<br />
hyopneumoniae have been described: p65, p50 <strong>and</strong> p44 (Wise <strong>and</strong> Kim, 1987). The<br />
recently sequenced genome <strong>of</strong> M. hyopneumoniae revealed 53 open reading frames<br />
with prokaryotic lipoprotein lipid attachment sites, but only 3 <strong>of</strong> them have a<br />
functional assignment (Minion et al., 2004). P46 <strong>and</strong> P65 have already been studied<br />
<strong>and</strong> found to be strong species-specific immunogenic proteins (Futo et al., 1995;<br />
Schmidt et al., 2004). P65 has lipolytic properties (Schmidt et al., 2004). The<br />
presence <strong>of</strong> TLR2 <strong>and</strong> TLR6 on porcine alveolar macrophages was demonstrated by<br />
Muneta et al. (2003). The same authors also demonstrated that blocking TLR2 <strong>and</strong><br />
TLR6 by monoclonal antibodies resulted in a decrease <strong>of</strong> TNF-α production by<br />
alveolar macrophages in vitro. The blocking did not completely inhibit TNF-α<br />
production, suggesting that other receptors may intervene in cytokine production<br />
(Muneta et al., 2003).<br />
Several studied mycoplasmas have the tendency to persist for long periods at<br />
the site <strong>of</strong> infection despite <strong>of</strong> the presence <strong>of</strong> a strong immune response (Adegboye,<br />
1978). M. hyopneumoniae has been demonstrated in pig lungs until 185 days after<br />
infection (Fano et al., 2004). The ability to polyclonally activate cells <strong>of</strong> the immune<br />
system may be one possible mechanism to modulate immunity <strong>and</strong> avoid clearance<br />
(Simecka, 2005). Another approach that mycoplasma may use to avoid clearance is<br />
through the variation <strong>of</strong> cell surface antigens. The best known mechanism is<br />
phenotypic plasticity by antigenic variation (Rottem, 2003). The number <strong>of</strong> genes<br />
involved in diversifying the antigenic nature <strong>of</strong> their cell is relatively high compared<br />
to other bacteria (Razin et al., 1998). The system used for this surface variation has<br />
been described in M. hyorhinis, a gene family called vlp genes, in M. gallisepticum<br />
(pMGA genes), M. bovis (vsp genes) <strong>and</strong> some human mycoplasmas (Raizin et al.,<br />
1998). Mostly lipoproteins are involved. Genes for lipoproteins contain t<strong>and</strong>em<br />
repeats that undergo slipped str<strong>and</strong> mispairing, resulting in either phase switching or<br />
phase variation. A similar system has not (yet) been described for M. hyopneumoniae.<br />
Compared to other Mycoplasmas spp., M. hyopneumoniae has few significant t<strong>and</strong>em<br />
repeat sequences, with one exception, the gene for the cilium adhesin P97, as<br />
described earlier (Djordjevic et al., 2004; Minion et al., 2004).<br />
- 20 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
REFERENCES<br />
1. Adegboye, D.S. (1978). A re<strong>view</strong> <strong>of</strong> Mycoplasma-induced immunosuppression. British Veterinary<br />
Journal 134, 556-560.<br />
2. Akira, S. & Hemmi, H. (2003). Recognition <strong>of</strong> pathogen-associated molecular patterns by TLR<br />
family. Immunology Letters 85, 85-95.<br />
3. Asai, T., Okada, M., Ono, M., Irisawa, T., Mori, Y., Yokomizo, Y. & Sato, S. (1993).<br />
Increased levels <strong>of</strong> tumor necrosis factor <strong>and</strong> interleukin-1 in bronchoalveolar lavage fluids from<br />
pigs infected with Mycoplasma hyopneumoniae. Veterinary Immunology <strong>and</strong> Immunopathology<br />
38, 253-260.<br />
4. Asai, T., Okada, M., Ono, M., Mori, Y., Yokomizo, Y. & Sato, S. (1994). Detection <strong>of</strong><br />
interleukin-6 <strong>and</strong> prostagl<strong>and</strong>in E2 in bronchoalveolar lavage fluids <strong>of</strong> pigs experimentally<br />
infected with Mycoplasma hyopneumoniae. Veterinary Immunology <strong>and</strong> Immunopathology 44,<br />
97-102.<br />
5. Blanchard, B., Vena, M.M., Cavalier, A., Le Lannic, J., Gouranton, J. & Kobisch, M. (1992).<br />
Electron microscopic observation <strong>of</strong> the respiratory tract <strong>of</strong> SPF piglets inoculated with<br />
Mycoplasma hyopneumoniae. Veterinary Microbiology 30, 329-341.<br />
6. Caruso, J.P. & Ross, R.F. (1990). Effects <strong>of</strong> Mycoplasma hyopneumoniae <strong>and</strong> Actinobacillus<br />
(Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine.<br />
American Journal <strong>of</strong> Veterinary Research 51, 227-231.<br />
7. Chambaud, I., Wróblewski, H. & Blanchard, A. (1999). Interactions between mycoplasma<br />
lipoproteins <strong>and</strong> the host immune system. Trends in Microbiology 7, 493-499.<br />
8. Chen, J.R., Lin, J.H., Weng, C.N. & Lai, S.S. (1998). Identification <strong>of</strong> a novel adhesion-like<br />
glycoprotein from Mycoplasma hyopneumoniae. Veterinary Microbiology 62, 97-110.<br />
9. Christensen, G., Sørensen, V. & Mousing, J. (1999). Diseases <strong>of</strong> the respiratory system. In:<br />
Straw, B.E., D’Allaire, S., Mengeling, W.L., Taylor, D.J. (Editors), Diseases <strong>of</strong> swine, 8 th edition,<br />
Iowa State University Press, Ames, Iowa, pp. 913-940.<br />
10. Cole, B.C. (1991). The immunobiology <strong>of</strong> Mycoplasma arthritidis <strong>and</strong> its superantigen MAM.<br />
Current Topics in Microbiology <strong>and</strong> Immunology 174, 107-119.<br />
11. Darai, G., Flugel, R.M., Zoller, L., Matz, B., Kreig, A., Gelderblom, H., Delius, H. & Leach,<br />
R.H. (1981). The plaque-forming factor for mink lung cells present in cytomegalovirus <strong>and</strong><br />
herpes-zoster virus stocks identified as Mycoplasma hyorhinis. The Journal <strong>of</strong> General Virology<br />
55, 201-205.<br />
12. Debey, M.C. & Ross, R.F. (1994). Ciliostasis <strong>and</strong> loss <strong>of</strong> cilia induced by Mycoplasma<br />
hyopneumoniae in porcine tracheal organ cultures. Infection <strong>and</strong> Immunity 62, 5312-5318.<br />
13. Deiters, U. & Mülhradt, P.F. (1999). Mycoplasmal lipopeptide MALP-2 induced the<br />
chemoattractant proteins macrophage inflammatory protein 1α (MIP-1α), monocyte<br />
chemoattractant protein 1, <strong>and</strong> MIP-2 <strong>and</strong> promotes leukocyte infiltration in mice. Infection <strong>and</strong><br />
Immunity 67, 3390-3398.<br />
14. Djordjevic, S.P., Cordwell, S.J., Djordjevic, M.A., Wilton, J. & Minion, F.C. (2004). Proteolytic<br />
processing <strong>of</strong> the Mycoplasma hyopneumoniae cilium adhesion. Infection <strong>and</strong> Immunity 72, 2791-<br />
2802.<br />
- 21 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
15. Djordjevic, S.P., Eamens, G.J., Romalis, L.F., Nicholls, P.J., Taylor, V. & Chin, J. (1997).<br />
Serum <strong>and</strong> mucosal antibody responses <strong>and</strong> protection in pigs vaccinated against Mycoplasma<br />
hyopneumoniae with vaccines containing a denatured membrane antigen pool <strong>and</strong> adjuvant.<br />
Australian Veterinary Journal 75, 504-511.<br />
16. Fagan, P.K., Walker, M.J., Chin, J., Eamens, G.J. & Djordjevic S.P. (2001). Oral<br />
immunisation <strong>of</strong> swine with attenuated Salmonella typhimurium aroA SL3261 expressing a<br />
recombinant antigen <strong>of</strong> Mycoplasma hyopneumoniae (NrdF) primes the immune system for a NrdF<br />
specific secretory IgA response in the lungs. Microbial Pathogenesis 30, 101-110.<br />
17. Fano, E., Pijoan, C. & Dee, S. (2004). Assessing the duration <strong>of</strong> Mycoplasma hyopneumoniae<br />
infection in gilts. Proceedings <strong>of</strong> the International Pig Veterinary Society, 18 th Congress, June 27-<br />
Juli 1, Hamburg / Germany, pp. 167.<br />
18. Futo, S., Seto, Y., Okada, M., Sato, S., Suzuki, T., Kawai, K., Imada, Y. & Mori, Y. (1995).<br />
Recombinant 46-kilodalton surface antigen (P46) <strong>of</strong> Mycoplasma hyopneumoniae expressed in<br />
Escherichia coli can be used for early specific diagnosis <strong>of</strong> mycoplasmal pneumonia <strong>of</strong> swine by<br />
enzyme-linked immunosorbent assay. Journal <strong>of</strong> Clinical Microbiology 33, 680-683.<br />
19. Gabridge, M.G. & Murphy, W.H. (1971). Toxic membrane fractions from Mycoplasma<br />
fermentans. Infection <strong>and</strong> Immunity 4, 678-682.<br />
20. Gabridge, M.G. & Schneider, P.R. (1975). Cytotoxic effect <strong>of</strong> Mycoplasma fermentans on<br />
mouse thymocytes. Infection <strong>and</strong> Immunity 11, 460-465.<br />
21. Geary, S.J. & Walczak, E.M. (1985). Isolation <strong>of</strong> a cytopathic factor from Mycoplasma<br />
hyopneumoniae. Infection <strong>and</strong> Immunity 48, 576-578.<br />
22. Henderson, B., Poole, S. & Wilson, M. (1996). Bacterial modulins: a novel class <strong>of</strong> virulence<br />
factors which cause host tissue pathology by inducing cytokine synthesis. Microbiological re<strong>view</strong>s<br />
60, 316-341.<br />
23. Hodge, L.M. & Simecka, J.W. (2002). Role <strong>of</strong> upper <strong>and</strong> lower respiratory tract immunity in<br />
resistance to Mycoplasma respiratory disease. The Journal <strong>of</strong> Infectious Diseases 186, 290-294.<br />
24. Hsu, T., Artiushin, S. & Minion, F.C. (1997). Cloning <strong>and</strong> analysis <strong>of</strong> P97, a respiratory cilium<br />
adhesion gene <strong>of</strong> Mycoplasma hyopneumoniae. Journal <strong>of</strong> Bacteriology 179, 1317-1323.<br />
25. Hsu, T. & Minion, F.C. (1998a). Molecular analysis <strong>of</strong> the P97 cilium adhesin operon <strong>of</strong><br />
Mycoplasma hyopneumoniae. Gene 214, 13-23.<br />
26. Hsu, T. & Minion, F.C. (1998b). Identification <strong>of</strong> the cilium binding epitope <strong>of</strong> the Mycoplasma<br />
hyopneumoniae P97 adhesin. Infection <strong>and</strong> Immunity 66, 4762-4766.<br />
27. Huang, Y.T., Chu, R.M., Liu, R.S. & Weng, C.N. (1990). Morphologic studies <strong>of</strong><br />
intrapulmonary airway mucosa-associated lymphoid tissues in swine. Veterinary Immunology <strong>and</strong><br />
Immunopathology 25, 13.22.<br />
28. Into, T., Kiura, K., Yasuda, M., Kataoka, H., Inoue, N., Hasebe, A., Takeda, K., Akira, S. &<br />
Shibata, K. (2004). Stimulation <strong>of</strong> human Toll-like receptor (TLR)2 <strong>and</strong> TLR6 with membrane<br />
lipoproteins <strong>of</strong> Mycoplasma fermentans induces apoptotic cell death after NF-κB activation.<br />
Cellular Microbiology 6, 187-199.<br />
29. Jones, H., Tabor, L., Sun, X., Woolard, M. & Simecka, J. (2002). Depletion <strong>of</strong> CD8+ cells<br />
exacerbates CD4+ Th-cell associated inflammatory lesions during murine mycoplasma respiratory<br />
disease. Journal <strong>of</strong> Immunology 168, 3493-3501.<br />
- 22 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
30. Kwon, D. & Chae, C. (1999). Detection <strong>and</strong> localization <strong>of</strong> Mycoplasma hyopneumoniae DNA in<br />
lungs from naturally infected pigs by in situ hybridization using a digoxigenin-labeled probe.<br />
Veterinary Pathology 36, 308-313.<br />
31. Livingston, D.W., Stair, E.L., Underdahl, N.R. & Mebus, C.A. (1972). Pathogenesis <strong>of</strong><br />
Mycoplasma pneumonia in swine. American Journal <strong>of</strong> Veterinary Research 33, 2249-2258.<br />
32. Loveless, R.W. & Feizi, T. (1989). Sialo-oligosaccharide receptors for Mycoplasma pneumoniae<br />
<strong>and</strong> related oligosaccharides <strong>of</strong> poly-N-acetyl lactosamine series are polarized at the cilia <strong>and</strong><br />
apical microvillar domains <strong>of</strong> the ciliated cells in human bronchial epithelium. Infection <strong>and</strong><br />
Immunity 57, 1285-1289.<br />
33. Lührmann, A., Deiters, U., Skokowa, J., Hanke, M., Gessner, J.E., Mülhradt, P.F. Pabst, R.<br />
& Tschernig, T. (2002). In vivo effects <strong>of</strong> a synthetic 2-kilodalton macrophage activating<br />
lipopeptide <strong>of</strong> Mycoplasma fermentans after pulmonary application. Infection <strong>and</strong> Immunity 70,<br />
3785-3792.<br />
34. Mebus, C.A. & Underdahl, N.R. (1977). Scanning electron microscopy <strong>of</strong> trachea <strong>and</strong> bronchi<br />
from gnotobiotic pigs inoculated with Mycoplasma hyopneumoniae. American Journal <strong>of</strong><br />
Veterinary Research 38, 1249-1254.<br />
35. Messier, S., Ross, R.F. & Paul, P.S. (1990). Humoral <strong>and</strong> cellular immune responses <strong>of</strong> pigs<br />
inoculated with Mycoplasma hyopneumoniae. American Journal <strong>of</strong> Veterinary Research 51, 52-58.<br />
36. Minion, F.C., Adams, C. & Hsu, T. (2000). R1 region on P97 mediates adherence <strong>of</strong><br />
Mycoplasma hyopneumoniae to swine cilia. Infection <strong>and</strong> Immunity 68, 3056-3060.<br />
37. Minion, F.C., Lefkowitz, E.J., Madsen, M.L., Cleary, B.J., Swartzell, S.M. & Mahairas, G.G.<br />
(2004). The genome sequence <strong>of</strong> Mycoplasma hyopneumoniae strain 232, the agent <strong>of</strong> swine<br />
mycoplasmosis. Journal <strong>of</strong> Bacteriology 186, 7123-7133.<br />
38. Mühlradt, P.F., Kieβ, M., Meyer, H., Süβmuth, R. & Jung, G. (1997). Isolation, structure<br />
elucidation, <strong>and</strong> synthesis <strong>of</strong> a macrophage stimulatory lipopeptide from Mycoplasma fermentans<br />
acting at picomolar concentration. The Journal <strong>of</strong> Experimental Medicine 185, 1951-1958.<br />
39. Mühlradt, P.F., Kieβ, M., Meyer, H., Süβmuth, R. & Jung, G. (1998). Structure <strong>and</strong> specific<br />
activity <strong>of</strong> macrophage-stimulating lipopeptides from Mycoplasma hyorhinis. Infection <strong>and</strong><br />
Immunity 66, 4804-4810.<br />
40. Muneta, Y., Uenishi, H., Kikuma, R., Yoshihara, K., Shimoji, Y., Yamamoto, R.,<br />
Hamashima, N., Yokomizo, Y. & Mori, Y. (2003). Porcine TLR2 <strong>and</strong> TLR6: Identification <strong>and</strong><br />
their involvement in Mycoplasma hyopneumoniae infection. Journal <strong>of</strong> Interferon & Cytokine<br />
Research 23, 583-590.<br />
41. Okada, M., Asai, T., Ono, M., Sakano, T. & Sato, S. (2000). Cytological <strong>and</strong> immunological<br />
changes in bronchoalveolar lavage fluid <strong>and</strong> histological observation <strong>of</strong> lung lesions in pigs<br />
immunized with Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture<br />
supernate. Vaccine 18, 2825-2831.<br />
42. Okusawa, T., Fujita, M., Nakamura, J., Into, T., Yasuda, M., Yoshimura, A., Hara, Y.,<br />
Hasebe, A., Golenbock, D.T., Morita, M., Kuroki, Y., Ogawa, T. & Shibata, K. (2004).<br />
Relationship between structures <strong>and</strong> biological activities <strong>of</strong> mycoplasmal diacylated lipopeptides<br />
<strong>and</strong> their recognition by Toll-like receptors 2 <strong>and</strong> 6. Infection <strong>and</strong> Immunity 72, 1657-1665.<br />
43. Park, S-C., Yibchok-Anun, S., Cheng, H., Young, T.F., Thacker, E.L., Minion, F.C., Ross,<br />
R.F. & Hsu, W.H. (2002). Mycoplasma hyopneumoniae increased intracellular calcium release in<br />
porcine ciliated tracheal cells. Infection <strong>and</strong> Immunity 70, 2502-2506.<br />
- 23 -
1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
44. Razin, S. & Jacobs, E. (1992). Mycoplasma adhesion. Journal <strong>of</strong> General Microbiology 138, 407-<br />
422.<br />
45. Razin, S., Yogev, D. & Naot, Y. (1998). Molecular biology <strong>and</strong> pathogenicity <strong>of</strong> Mycoplasmas.<br />
Microbiology <strong>and</strong> Molecular Biology Re<strong>view</strong>s 62, 1094-1156.<br />
46. Rodríguez, F., Ramírez, G.A., Sarradell, J., Andrada, M. & Lorenzo, H. (2004).<br />
Immunohistochemical labelling <strong>of</strong> cytokines in lung lesions <strong>of</strong> pigs naturally infected with<br />
Mycoplasma hyopneumoniae. Journal <strong>of</strong> Comparative Pathology 130, 306-312.<br />
47. Rottem, S. (2003). Interaction <strong>of</strong> Mycoplasmas with host cells. Physiological Re<strong>view</strong>s 83, 417-<br />
432.<br />
48. Sarradell, J., Andrada, M., Ramírez, A.S., Fernández, A., Gómez-Villam<strong>and</strong>os, J.C., Jover,<br />
A., Lorenzo, H., Herráez, P. & Rodríguez, F. (2003). A morphological <strong>and</strong><br />
immunohistochemical study <strong>of</strong> the bronchus-associated lymphoid tissue <strong>of</strong> pigs naturally infected<br />
with Mycoplasma hyopneumoniae. Veterinary Pathology 40, 395-404.<br />
49. Schmidt, J.A., Browning, G.F. & Markham, P.F. (2004). Mycoplasma hyopneumoniae p65<br />
surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids. Journal <strong>of</strong><br />
Bacteriology 186, 5790-5798.<br />
50. Sheldrake, R.F. (1990). IgA immune responses in the respiratory tract <strong>of</strong> pigs. Research in<br />
Veterinary Science 49, 98-103<br />
51. Sheldrake, R.F., Romalis, L.F. & Saunders, M.M. (1993). Serum <strong>and</strong> mucosal antibody<br />
responses against Mycoplasma hyopneumoniae following intraperitoneal vaccination <strong>and</strong><br />
challenge <strong>of</strong> pigs with M. hyopneumoniae. Research in Veterinary Science 55, 371-376.<br />
52. Shibata, K., Hasebe, A., Into, T., Yamada, M. & Watanabe, T. (2000). The N-terminal<br />
lipopeptide <strong>of</strong> a 44-kDa membrane-bound lipoprotein <strong>of</strong> Mycoplasma salivarum is responsible for<br />
the expression <strong>of</strong> intercellular adhesion molecule-1 on the cell surface <strong>of</strong> normal human gingival<br />
fibroblasts. The Journal <strong>of</strong> Immunology 165, 6538-6544.<br />
53. Simecka, J.W. (2005). Immune responses following Mycoplasma infection. In: Mycoplasmas:<br />
molecular biology, pathogenicity <strong>and</strong> strategies for control. Blanchard, A., Browning, G., Editors.<br />
Horizon Bioscience, Norfolk, UK, pp. 485-534.<br />
54. Simecka, J.W., Ross, S.E., Cassell, G.H. & Davis, J.K. (1993). Interactions <strong>of</strong> Mycoplasmas<br />
with B cells: antibody production <strong>and</strong> nonspecific effects. Clinical Infectious Diseases, 17 (Suppl.<br />
1), S176-182.<br />
55. Tajima, M. & Yagihashi, T. (1982). Interaction <strong>of</strong> Mycoplasma hyopneumoniae with the porcine<br />
respiratory epithelium as observed by electron microscopy. Infection <strong>and</strong> Immunity 37, 1162-<br />
1169.<br />
56. Tajima, M., Yagihashi, T. & Nunoya, T. (1985). Ultrastructure <strong>of</strong> mycoplasmal capsule as<br />
revealed by stabilization with antiserum <strong>and</strong> staining with ruthenium red. The Japanese Journal <strong>of</strong><br />
Veterinary Science 47, 217-223.<br />
57. Thanawongnuwech, R., Young, T.F., Thacker, B.J. & Thacker, E.L. (2001). Differential<br />
production <strong>of</strong> proinflammatory cytokines: in vitro PRRSV <strong>and</strong> Mycoplasma hyopneumoniae coinfection<br />
model. Veterinary Immunology <strong>and</strong> Immunopathology 79, 115-127.<br />
58. Thomas, A., Sachse, K., Dizier, I., Grajetzki, C., Farnir, F., Mainil, J.G. & Linden, A. (2003).<br />
Adherence to various host cell lines <strong>of</strong> Mycoplasma bovis strains differing in pathogenic <strong>and</strong><br />
cultural features. Veterinary Microbiology 91, 101-113.<br />
59. Tully, J.G. (1981). Mycoplasmal toxins. Israel Journal <strong>of</strong> Medical Sciences 17, 604-607.<br />
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1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA<br />
60. Walker, J., Lee, R., Mathy, N., Doughty, S. & Conlon, J. (1996). Restricted B-cell responses to<br />
microbial challenge <strong>of</strong> the respiratory tract. Veterinary Immunology <strong>and</strong> Immunopathology 54,<br />
197-204.<br />
61. Wise, K. & Kim, M. (1987). Major membrane surface proteins <strong>of</strong> Mycoplasma hyopneumoniae<br />
selectively modified by covalently bound lipid. Journal <strong>of</strong> Bacteriology 169, 5546-5555.<br />
62. Zhang, Q., Young, T.F. & Ross, R.F. (1994). Glycolipid receptors for attachement <strong>of</strong><br />
Mycoplasma hyopneumoniae to porcine respiratory ciliated cells. Infection <strong>and</strong> Immunity 62,<br />
4367-4373.<br />
63. Zhang, Q., Young, T.F. & Ross, R.F. (1995). Identification <strong>and</strong> characterization <strong>of</strong> a<br />
Mycoplasma hyopneumoniae adhesion. Infection <strong>and</strong> Immunity 63, 1013-1019.<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF<br />
ENZOOTIC PNEUMONIA IN PIGS<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
Enzootic pneumonia is responsible for large economic losses in the pig<br />
industry worldwide. This makes prevention or treatment <strong>of</strong> the disease inevitable to<br />
limit the losses. Control <strong>of</strong> mycoplasmal infections can be accomplished by<br />
optimizing <strong>of</strong> management <strong>and</strong> housing, by vaccination <strong>and</strong> by preventive medication.<br />
Improvement <strong>of</strong> the housing <strong>and</strong> management conditions (ventilation, stocking<br />
density, purchase policy <strong>and</strong> hygiene) are primordial in the control <strong>of</strong> enzootic<br />
pneumonia <strong>and</strong> should be the first to be accomplished. In a well managed herd still<br />
suffering from M. hyopneumoniae infections, vaccination is a further important tool to<br />
reduce economic losses. Vaccination against M. hyopneumoniae in pigs is<br />
successfully practiced in most countries with an intensive pig production (Maes et al.,<br />
1998; 1999; 2003; Haesebrouck et al., 2004). Local, mucosal, humoral <strong>and</strong> cellular<br />
immune responses are induced, resulting in a reduction <strong>of</strong> lung lesions (Thacker et al.,<br />
2000). Economical benefit is achieved by an increase in daily weight gain (Jensen et<br />
al., 2002), a decrease <strong>of</strong> the feed conversion ratio <strong>and</strong> a reduction in medication costs<br />
(Maes et al., 2003).<br />
Nevertheless, the use <strong>of</strong> antimicrobials, either in a preventive or curative way<br />
remains necessary in many pig herds, due to a non-vaccination policy, vaccination<br />
failure or purchase <strong>of</strong> piglets with unknown vaccination status or from several<br />
suppliers. The control <strong>of</strong> the disease by medication can be approached by strategic<br />
administration <strong>of</strong> antimicrobials. This paper gives an over<strong>view</strong> <strong>of</strong> antimicrobials<br />
active against M. hyopneumoniae <strong>and</strong> summarizes in vitro <strong>and</strong> in vivo studies<br />
conducted to evaluate that activity.<br />
ANTIMICROBIAL AGENTS USED FOR THE TREATMENT OF M.<br />
HYOPNEUMONIAE INFECTIONS<br />
Potentially active antimicrobials against M. hyopneumoniae include<br />
tetracyclines, macrolides <strong>and</strong> lincosamides, pleuromutilins, fluoroquinolones,<br />
florfenicol, aminoglycosides <strong>and</strong> aminocyclitols. To control <strong>and</strong> treat respiratory<br />
disease in swine, tetracyclines <strong>and</strong> macrolides are most frequently used (Chauvin et<br />
al., 2002; Timmerman et al., 2005). Only fluoroquinolones <strong>and</strong> aminoglycosides have<br />
mycoplasmacidal effect (Hannan et al., 1989) which is an important characteristic<br />
when eradication <strong>of</strong> enzootic pneumonia is intended.<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
In Table 1, recommended systemic or oral dosages <strong>of</strong> antimicrobials used to<br />
treat Mycoplasma hyopneumoniae infections are summarized.<br />
Table 1: Recommended systemic / oral dosages <strong>of</strong> antimicrobials used to treat<br />
Mycoplasma hyopneumoniae infections in pigs.<br />
Type <strong>of</strong><br />
antimicrobial<br />
Route <strong>of</strong><br />
administration<br />
Dose (mg/kg<br />
bodyweight)<br />
Interval (h)<br />
Oxytetracycline IM<br />
Oral<br />
10 - 20<br />
20-50<br />
12 - 24<br />
-<br />
Doxycyline hyclate Oral 10<br />
Lincomycin<br />
hydrochloride<br />
IM<br />
Oral<br />
10<br />
5-10<br />
24<br />
-<br />
Tilmicosin Oral 8-20 -<br />
Tylosin<br />
IM<br />
Oral<br />
5-20<br />
30-60<br />
24<br />
-<br />
Tulathromycin IM 2.5 -<br />
Tiamulin Oral 8.8 -<br />
Valnemulin Oral 3-4 -<br />
Florfenicol IM 15 48<br />
Enr<strong>of</strong>loxacin IM 2.5-5 24<br />
Marb<strong>of</strong>loxacin IM 2 24<br />
Flumequin Oral 15 -<br />
Gentamicin IM 3-5 24<br />
Spectinomycin IM 20 12<br />
1. Tetracyclines<br />
Tetracyclines (including tetracycline, chlortetracycline, doxycycline, <strong>and</strong><br />
oxytetracycline) are amphoteric molecules that are poorly soluble in water at pH 7.<br />
Tetracyclines are bacteriostatic antibiotics that bind irreversibly to receptors <strong>of</strong> the<br />
30S bacterial ribosome, where they interfere with the association <strong>of</strong> aminoacyl-tRNA<br />
to the acceptor site on the mRNA ribosome complex resulting in inhibition <strong>of</strong><br />
bacterial protein synthesis (Prescott, 2000b; Chopra <strong>and</strong> Roberts, 2001). They are<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
broad-spectrum antimicrobials active against Gram-positive bacteria, including<br />
mycoplasmas, <strong>and</strong> Gram-negative bacteria, including chlamydiae <strong>and</strong> rickettsias, <strong>and</strong><br />
some protozoa (Babesia, Theileria). In the host, their bacteriostatic activity is optimal<br />
in an acid environment. Oral bioavailability is lowered when administered in feed,<br />
except for doxycycline. Bivalent cations (calcium, magnesium, iron) decrease the<br />
absorption <strong>and</strong> activity by chelating tetracycline (Luthman <strong>and</strong> Jacobsson, 1983).<br />
Also, when administered via the drinking water, the bioavailability can be influenced<br />
by the water quality (degree <strong>of</strong> acidity) <strong>and</strong> bivalent cations in the material (iron) <strong>of</strong><br />
the water pipes (Prescott, 2000b). Tetracyclines are lipophilic <strong>and</strong> therefore diffuse<br />
easily through biological barriers <strong>and</strong> cell membranes. Generally speaking,<br />
tetracyclines are the drugs <strong>of</strong> first choice for the treatment <strong>of</strong> respiratory Mycoplasma<br />
infections in pigs.<br />
2. Macrolides <strong>and</strong> lincosamides<br />
Macrolides <strong>and</strong> lincosamides are structurally distinct antibiotics but share<br />
many common properties such as a similar mode <strong>of</strong> action <strong>and</strong> an overlapping binding<br />
site on the 50S ribosome (Weisblum, 1998; Vester <strong>and</strong> Douthwaite, 2001). They are<br />
basic compounds, have a high lipid solubility <strong>and</strong> have a good absorption from the<br />
intestine <strong>and</strong> tissue distribution. After uptake, macrolides <strong>and</strong> lincosamides<br />
accumulate mainly in phagocytes, with 51 - 85 % localized in lysozomes (Scorneaux<br />
<strong>and</strong> Shryock, 1998). Like tetracyclines, macrolides <strong>and</strong> lincosamides inhibit protein<br />
synthesis at the ribosome level <strong>and</strong> are bacteriostatic. By binding to the 50S subunit <strong>of</strong><br />
the ribosome, they block the growth <strong>of</strong> the nascent peptide chain, probably causing<br />
premature dissociation <strong>of</strong> the peptidyl-tRNA from the ribosome (Vester <strong>and</strong><br />
Douthwaite, 2001). Macrolides <strong>and</strong> lincosamides have been mapped by chemical<br />
footprinting to the central loop in domain V <strong>of</strong> 23S rRNA, which is associated with<br />
the peptidyl transferase activity (Cundliffe, 1990).<br />
Macrolides are divided into three subgroups according to the size <strong>of</strong> their<br />
lactone ring. Erythromycin <strong>and</strong> ole<strong>and</strong>omycin belong to the 14-membered ring<br />
macrolides, while josamycin, kitasamycin, tylosin, tilmicosin <strong>and</strong> spiramycin belong<br />
to the 16-membered ring subgroup. Azithromycin <strong>and</strong> tulathromycin are members <strong>of</strong><br />
the 15 membered ring macrolides. Macrolides are active against Gram-positive<br />
bacteria (Rhodococcus equi, Bacillus spp., Corynebacterium spp., Erysipelothrix<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
rhusiopathiae, Listeria spp., Staphylococcus spp., Streptococcus spp., Mycoplasma<br />
spp.), selected Gram-negative bacteria (Pasteurella, Mannheimia, Leptospira,<br />
Campylobacter, Actinobacillus) <strong>and</strong> anaerobes (Actinomyces spp., Clostridium spp.,<br />
Bacteroides spp., Prevotella spp., …).<br />
Lincosamides (lincomycin <strong>and</strong> clindamycin) are active against many Grampositive<br />
bacteria <strong>and</strong> anaerobes but they are considerably less effective against Gramnegative<br />
bacteria <strong>and</strong> Mycoplasma spp. compared to macrolides (Prescot, 2000c).<br />
Absorption <strong>of</strong> lincomycin <strong>and</strong> clindamycin is very good after oral administration in<br />
monogastric animals. Absorption is slower when administered together with food.<br />
Lincosamides diffuse well through biological barriers <strong>and</strong> can obtain high tissue<br />
concentrations.<br />
3. Pleuromutilins<br />
Pleuromutilins (tiamulin <strong>and</strong> valnemulin) share many common properties such<br />
as a similar mode <strong>of</strong> action <strong>and</strong> an overlapping binding site on the 50S ribosome<br />
(Weisblum, 1998; Vester <strong>and</strong> Douthwaite, 2001) with macrolides <strong>and</strong> lincosamides.<br />
They are bacteriostatic, have a comparable antimicrobial spectrum as macrolides, but<br />
have remarkable activities against anaerobes <strong>and</strong> mycoplasmas. Pleuromutilins are<br />
exclusively used in veterinary medicine, mainly in swine. Valnemulin is 30 times<br />
more active than tiamulin, especially against Mycoplasma spp. (Aitken et al., 1999).<br />
Pleuromutilins are almost completely absorbed after oral administration in<br />
monogastrates (Prescott, 2000c). The bioavailability <strong>of</strong> both compounds reaches over<br />
90% <strong>and</strong> both achieve high drug concentrations in the lung (Burch, 2001).<br />
4. Fluoroquinolones<br />
Fluoroquinolones are widely used broad-spectrum antibiotics in human <strong>and</strong><br />
veterinary medicine. Nalidixic acid, the original non-fluorinated molecule <strong>of</strong> the<br />
quinolone class, is inactive against Gram-positive bacteria <strong>and</strong> Mycoplasma spp.<br />
(Roberts, 1992). These micro-organisms are however susceptible to the derived<br />
fluoroquinolone classes. Compared to flumequine, the fluoroquinolones enr<strong>of</strong>loxacin,<br />
dan<strong>of</strong>loxacin, cipr<strong>of</strong>loxacin <strong>and</strong> norfloxacin show an enhanced activity against all<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
mycoplasmas studied (Hannan et al., 1997a) <strong>and</strong> are mycoplasmacidal when they are<br />
administered in an increased dose (Arai et al., 1992).<br />
The intracellular targets <strong>of</strong> fluoroquinolones in bacteria are considered the<br />
type II topoisomerases, DNA gyrase <strong>and</strong> topoisomerase IV. Both enzymes are<br />
essential for DNA replication. DNA gyrase is composed <strong>of</strong> two GyrA <strong>and</strong> two GyrB<br />
subunits (A 2 B 2 ), encoded by the gyrA <strong>and</strong> gyrB genes, respectively. This tetrameric<br />
enzyme catalyzes ATP-dependent negative supercoiling <strong>of</strong> DNA. Topoisomerase IV,<br />
a C 2 E 2 tetramer encoded by the parC <strong>and</strong> parE genes, separates interlocked DNA<br />
str<strong>and</strong>s to allow segregation <strong>of</strong> daughter chromosomes into daughter cells. ParC is<br />
similar in structure to GyrA while ParE is homologous to GyrB (Hooper, 2002).<br />
In monogastric animals, orally administered fluoroquinolones are absorbed quickly<br />
<strong>and</strong> completely (80-100%) (Inui et al., 1998). Maximum plasma concentrations are<br />
reached one hour after intake. Fluoroquinolones are strongly lipophilic <strong>and</strong> therefore a<br />
high tissue distribution is reached with good penetration through biological barriers.<br />
5. Florfenicol<br />
Florfenicol is exclusively used in veterinary medicine. The compound is<br />
analogous to chloramphenicol but does not cause irreversible depression <strong>of</strong> the bone<br />
marrow <strong>and</strong> can therefore be used in food producing animals. Florfenicol is a strong<br />
inhibitor <strong>of</strong> the microbial protein synthesis through irreversible binding with the 50S<br />
subunit <strong>of</strong> the ribosomes, abolishing the activity <strong>of</strong> peptidyl transferase. The antibiotic<br />
is bacteriostatic <strong>and</strong> active against most Gram-positive (including Mycoplasma spp.)<br />
<strong>and</strong> Gram-negative bacteria (including rickettsia <strong>and</strong> chlamydia) <strong>and</strong> anaerobes.<br />
Pharmacokinetic data on florfenicol metabolism in the pig are scarce, but the<br />
properties are generally similar to chloramphenicol. This lipophilic drug has a wide<br />
tissue distribution. Florfenicol is absorbed quickly <strong>and</strong> completely after oral<br />
administration, rapidly distributed <strong>and</strong> eliminated slowly. Feed intake did not<br />
significantly change the pharmacokinetics <strong>of</strong> florfenicol after oral administration in<br />
pigs (Liu et al., 2003).<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
6. Aminoglycosides <strong>and</strong> aminocyclitols<br />
Aminoglycosides (streptomycin, neomycin, kanamycin, gentamicin,<br />
tobramycin, apramycin <strong>and</strong> amikacin) <strong>and</strong> aminocyclitols (spectinomycin) are a<br />
widely used group <strong>of</strong> broad-spectrum antimicrobials which are bactericidal <strong>and</strong> target<br />
the bacterial ribosome. They inhibit bacterial protein synthesis by binding on one<br />
(streptomycin) or different sites in the 16S rRNA <strong>of</strong> the 30S subunit <strong>and</strong> to some<br />
ribosomal proteins, decreasing the fidelity <strong>of</strong> translation (Kotra et al., 2000). Other<br />
effects <strong>of</strong> aminoglycosides include interference with the cellular electron transport<br />
system, induction <strong>of</strong> RNA breakdown, inhibition <strong>of</strong> translation, effects on DNA<br />
metabolism <strong>and</strong> damage to cell membranes (Prescott, 2000a). They are active against<br />
several Gram-positive (including Mycoplasma spp. <strong>and</strong> Mycobacterium spp.) <strong>and</strong><br />
many Gram-negative bacteria. Their action against Streptococcus spp. is limited. The<br />
activity decreases in an anaerobic, acid or purulent environment since penetration<br />
through the cell membrane is in part an active oxydative transport.<br />
Aminoglycosides are administered orally or parenterally. Resorption is limited<br />
in the gastro-intestinal tract <strong>and</strong> they bind poorly to plasma proteins (less than 25%).<br />
Distribution after parenteral administration is limited to the extracellular fluid since<br />
aminoglycosides in general are too polar to cross mammalian cell membranes by<br />
diffusion. Poor diffusibility can be attributed to their low degree <strong>of</strong> lipid solubility<br />
(Giroux et al., 1995). Parenterally administered aminoglycosides easily penetrate in<br />
lung parenchyma <strong>and</strong> bronchial secretions according to the plasma-tissue<br />
concentration ratio (Saux et al., 1986; Goldstein et al., 2002).<br />
ROUTE OF ANTIMICROBIAL ADMINISTRATION<br />
In modern pig herds, animals are mostly treated as a group <strong>and</strong> not as single<br />
individuals. Consequently, treatment occurs predominantly through in-feed or inwater<br />
medication because this is easier to apply <strong>and</strong> less stressful compared to<br />
parenteral medication. Usually, only pigs suffering from acute disease receive<br />
parenteral injections on the first 2-3 days <strong>of</strong> disease, <strong>and</strong> are usually further treated<br />
per os. Parenteral injection <strong>of</strong>ten results in the best response, particularly in the case<br />
<strong>of</strong> acute respiratory tract disease (Henry <strong>and</strong> Apley, 1999).<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
Achieving sufficiently high levels <strong>of</strong> the administered drug in the lung tissue is<br />
difficult when using oral medication. Even though in-feed medication is the most<br />
common method <strong>of</strong> administering antimicrobials, there are several disadvantages.<br />
This approach is rather wasteful since diseased but also healthy pigs receive<br />
medication. This can be justified by the knowledge that penmates are at-risk to<br />
become infected as well, especially in herds with a high stocking density. Sick pigs<br />
<strong>of</strong>ten have a suppressed appetite <strong>and</strong> therefore do not eat sufficient amounts <strong>of</strong> the<br />
drug to reach therapeutic levels in their body. Adaptation <strong>of</strong> the antimicrobial dose in<br />
the feed or water may overcome this problem. A close follow-up <strong>of</strong> the feed intake is<br />
required to achieve appropriate therapeutic levels <strong>and</strong> to avoid toxic effects due to<br />
overdosing. Some antimicrobial agents are poorly absorbed or are metabolized by the<br />
low pH in the stomach <strong>and</strong> may therefore not achieve therapeutic levels. The choice<br />
<strong>of</strong> a correct antimicrobial is indispensable. In-feed medication is especially suited for<br />
endemic or chronic diseases, such as enzootic pneumonia, since a quick<br />
administration <strong>of</strong> the medicated feed is not as important as for acute infections with<br />
eg. Actinobacillus pleuropneumoniae (Henry <strong>and</strong> Apley, 1999; Friendship, 2000).<br />
Pulse-medication is a variant <strong>of</strong> in-feed or in-water medication (Burch, 1990). A short<br />
period during which a therapeutic dose <strong>of</strong> antimicrobials is included in the feed is<br />
alternated with a longer period without medication. During the non-medication period<br />
pigs are able to develop an active immune response.<br />
In-water medication which can be administered more quickly is recommended<br />
for acute diseases, such as Actinobacillus pleuropneumoniae infections. Sick pigs<br />
<strong>of</strong>ten continue to drink, even when they refuse food. A major disadvantage is the<br />
spilling that <strong>of</strong>ten occurs by playing with the drinking nipples out <strong>of</strong> boredom. Some<br />
antimicrobials are unstable in water, poorly soluble due to the water pH or react with<br />
the water pipe material (e.g. tetracyclines with iron). Water intake <strong>of</strong> the piglets may<br />
greatly vary with the season; appropriate follow-up <strong>of</strong> the water intake is a<br />
prerequisite for achieving correct therapeutic doses (Henry <strong>and</strong> Apley, 1999).<br />
M. hyopneumoniae infections can be managed metaphylaxially or<br />
prophylaxially. Metaphylaxis means the use <strong>of</strong> medication with therapeutic levels <strong>of</strong><br />
drugs when some animals are clinically diseased while others are subclinically<br />
diseased or at high risk. Prophylaxis is the use <strong>of</strong> medication with therapeutic levels<br />
<strong>of</strong> drugs during high-risk periods for disease. A high-risk period in Western Europe<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
for achieving M. hyopneumoniae infection is usually at 10 weeks <strong>of</strong> age, after transfer<br />
<strong>of</strong> animals to the finishing houses (Léon et al., 2001).<br />
THE EFFICACY OF SEVERAL ANTIMICROBIALS AGAINST M. HYOPNEUMONIAE<br />
INFECTIONS UNDER EXPERIMENTAL AND / OR FIELD CONDITIONS.<br />
Several studies have been conducted to assess the efficacy <strong>of</strong> various<br />
antimicrobials used for the prevention <strong>and</strong> the treatment <strong>of</strong> M. hyopneumoniae<br />
infections. Comparison <strong>of</strong> these studies is complicated due to considerable variations<br />
in study design. An over<strong>view</strong> <strong>of</strong> the conclusions drawn from studies published in<br />
peer-re<strong>view</strong>ed journals is given in Table 2 (experimental studies) <strong>and</strong> Table 3 (field<br />
studies).<br />
From these tables it can be concluded that for most antimicrobials tested,<br />
economic parameters (daily weight gain <strong>and</strong> feed conversion ratio) were improved<br />
<strong>and</strong> lung lesions as well as clinical signs were decreased in treated animals.<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
Table 2: The effect <strong>of</strong> different antimicrobial regimens for treatment <strong>of</strong> experimental<br />
infections with Mycoplasma hyopneumoniae.<br />
Antimicrobial drug, dosage,<br />
scheme<br />
6-chloro analogue <strong>of</strong> Norfloxacin<br />
400 or 200 ppm<br />
Norfloxacin 100 ppm<br />
feed medication during 3 weeks<br />
starting 1 month after infection<br />
Tiamulin 100 or 200 ppm<br />
feed medication during 10 days<br />
starting 2 days before infection<br />
Tiamulin 50 mg/kg<br />
feed medication during 10 days<br />
starting 1 month after infection<br />
Tiamulin 240 ppm<br />
feed medication during 10 days<br />
starting 10 days after infection<br />
Tiamulin 60, 120, 180 ppm<br />
medication in drinking water<br />
during 10 days starting 11 days<br />
after infection<br />
Tylosin tartrate 50 mg/kg<br />
combined with Tiamulin 10<br />
mg/kg<br />
medication in drinking water<br />
during 10 days starting 14 days<br />
after infection<br />
Effects<br />
improvement <strong>of</strong> DWG <strong>and</strong> FCR<br />
improvement <strong>of</strong> lung lesions by 6-<br />
chloro analogue at 400 ppm only<br />
improvement <strong>of</strong> DWG <strong>and</strong> FCR<br />
lung lesions were not prevented<br />
M. hyopneumoniae could still be<br />
isolated<br />
improvement <strong>of</strong> DWG, clinical signs,<br />
lung lesions<br />
M. hyopneumoniae could still be<br />
isolated<br />
improvement <strong>of</strong> DWG, FCR, clinical<br />
signs, <strong>and</strong> macroscopic <strong>and</strong> microscopic<br />
lung lesions<br />
no beneficial effect on DWG, FCR,<br />
clinical signs, <strong>and</strong> macroscopic <strong>and</strong><br />
microscopic lung lesions<br />
improvement <strong>of</strong> macroscopic lung<br />
lesions<br />
M. hyopneumoniae could not be<br />
isolated<br />
less frequently secondary bacterial<br />
infections<br />
Reference<br />
Hannan <strong>and</strong> Goodwin,<br />
1990<br />
Schuller et al., 1977<br />
Goodwin, 1979<br />
Kobisch <strong>and</strong> Sibelle,<br />
1982<br />
Ross <strong>and</strong> Cox, 1988<br />
Hannan et al., 1982<br />
DWG: daily weight gain; FCR: feed conversion ratio<br />
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Table 3: The effect <strong>of</strong> different antimicrobial regimens for treatment <strong>of</strong> Mycoplasma<br />
hyopneumoniae infections in herds clinically affected by enzootic pneumonia.<br />
Treatment<br />
Effects<br />
Reference<br />
Enr<strong>of</strong>loxacin 5 mg/kg<br />
Per os during 3 weeks starting after birth<br />
followed by parenteral treatment during<br />
3 weeks every 3 days starting in nursery<br />
unit followed by parenteral treatment<br />
during one week every 2 days<br />
prevention <strong>of</strong> clinical signs Frank, 1989<br />
Marb<strong>of</strong>loxacin 3 mg/kg<br />
IM during 3 days<br />
decrease <strong>of</strong> rectal temperature,<br />
improved DWG <strong>and</strong> clinical signs<br />
Thomas et al.,<br />
2000<br />
Chlortetracycline 800 ppm<br />
feed medication during 3 weeks<br />
Chlortetracycline 110 ppm<br />
feed medication for 1, 2, 3 or 4 months<br />
improvement <strong>of</strong> clinical signs <strong>and</strong><br />
oxygen saturation<br />
no improvement <strong>of</strong> DWG, FCR <strong>and</strong><br />
macroscopic lesions<br />
Ganter, 1995<br />
Ice et al., 1999<br />
Doxycycline 11 mg/kg<br />
feed medication during 8 days in the<br />
fattening unit<br />
improvement <strong>of</strong> DWG, incidence <strong>of</strong><br />
diseased pigs <strong>and</strong> cure rate<br />
Bousquet et al.,<br />
1998<br />
Lincomycin 5 mg/kg<br />
parenteral at day 1, 2, 3, <strong>and</strong> at day 39,<br />
40, 41<br />
Lincomycin 220 ppm<br />
feed medication during 3 weeks<br />
improved DWG, FCR, <strong>and</strong> clinical signs<br />
no significant improvements<br />
Lukert <strong>and</strong><br />
Mulkey, 1982<br />
Mateusen et al.,<br />
2002<br />
Tiamulin 200 ppm<br />
feed medication during 10 days in the<br />
growing unit<br />
Tiamulin 30 ppm<br />
feed medication during 8 weeks in pigs<br />
from 30 to 70 kg<br />
Tiamulin 100 ppm combined with<br />
Chlortetracycline 300 ppm or<br />
Chlortetracycline 300 ppm alone<br />
feed medication during 7 days<br />
improvement <strong>of</strong> DWG, clinical signs,<br />
macroscopic lung lesions, <strong>and</strong> mortality<br />
rate<br />
improvement <strong>of</strong> DWG, FCR<br />
no decrease in macroscopic lung lesions<br />
improvement <strong>of</strong> DWG, FCR, <strong>and</strong><br />
no improvement <strong>of</strong> macroscopic lung<br />
lesions for the combined group<br />
improvement <strong>of</strong> DWG for the single<br />
group only<br />
Martineau, 1980<br />
Burch, 1984<br />
Burch et al., 1986<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
Tylosin 4 mg/kg or<br />
Lincomycin 5 mg/kg<br />
parenteral during 3 days after birth <strong>and</strong><br />
for 3 days at weaning<br />
Tilmicosin 300 ppm in feed<br />
during 9 or 14 days<br />
Tilmicosin 200 ppm in feed<br />
during 3 weeks after weaning (days 34-<br />
55) <strong>and</strong> during 2 weeks in the nursery<br />
period (days 77-98)<br />
improved DWG with Tylosin<br />
no significant improvement <strong>of</strong> FCR <strong>and</strong><br />
clinical signs with both antibiotics<br />
improved DWG, clinical signs<br />
less secondary bacteria<br />
Beneficial effects <strong>of</strong> DWG, FCR,<br />
mortality, <strong>and</strong> macroscopic lesions<br />
comparable to M. hyopneumoniae<br />
vaccination<br />
Kunesh, 1981<br />
Binder et al., 1993<br />
Mateusen et al.,<br />
2001<br />
Tiamulin 200 ppm in feed<br />
Chlortetracycline 600 ppm in feed<br />
pulse medication:<br />
2 days treatment/2 weeks during the<br />
fattening period<br />
Tiamulin 30 ppm in feed<br />
Oxytetracycline 300 ppm in feed<br />
pulse medication:<br />
2 or 3 days treatment/week during the<br />
fattening period<br />
Tiamulin 40 ppm in feed<br />
Oxytetracycline 300 ppm in feed<br />
pulse medication:<br />
2 days treatment/week during the<br />
fattening period<br />
Tiamulin 100 ppm in feed<br />
during 7 day at weaning <strong>and</strong><br />
during 7 days at 4 months <strong>of</strong> age (<strong>and</strong><br />
during 7 days at 6 months <strong>of</strong> age)<br />
lower prevalence <strong>of</strong> macroscopic lung<br />
lesions<br />
no influence on severity <strong>of</strong> macroscopic<br />
lung lesions<br />
improved DWG <strong>and</strong> FCR, prevalence<br />
<strong>and</strong> severity <strong>of</strong> lung lesions<br />
financial benefit<br />
improved DWG <strong>and</strong> mortality rate<br />
no improvement <strong>of</strong> FCR <strong>and</strong> severity <strong>of</strong><br />
lung lesions<br />
Improved DWG <strong>and</strong> mortality rate,<br />
improvement <strong>of</strong> FCR <strong>and</strong> severity <strong>of</strong><br />
lung lesions, financial benefit<br />
Le Gr<strong>and</strong> <strong>and</strong><br />
Kobisch, 1996<br />
Kavanagh, 1994<br />
Jouglar et al., 1993<br />
Stipkovits et al.,<br />
2003<br />
DWG: daily weight gain; FCR: feed conversion ratio; IM: intramuscular<br />
ANTIMICROBIAL SUSCEPTIBILITY TESTING FOR MYCOPLASMA SPP.<br />
The Clinical <strong>and</strong> Laboratory St<strong>and</strong>ards Institute issues no specific guidelines<br />
for susceptibility testing <strong>of</strong> Mycoplasma spp. Therefore, there has been little<br />
st<strong>and</strong>ardization <strong>of</strong> the procedures used to determine the minimal inhibitory<br />
concentration (MIC) <strong>of</strong> antimicrobials against these micro-organisms. This makes the<br />
comparison <strong>of</strong> MIC results between laboratories difficult. St<strong>and</strong>ard broth <strong>and</strong> agar<br />
dilution methods used for susceptibility testing <strong>of</strong> other bacteria have been adapted for<br />
use with mycoplasmas. These methods, performed with appropriate controls, give<br />
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useful results for the treatment <strong>of</strong> mycoplasmal infections. Recently ‘Guidelines <strong>and</strong><br />
recommendations for antimicrobial minimum inhibitory concentration (MIC) testing<br />
against veterinary mycoplasma species’ were proposed by the ‘International Research<br />
Programme on Comparative Mycoplasmology (IRPCM)’ (Hannan, 2000). No single<br />
st<strong>and</strong>ardized medium or pH can be indicated for mycoplasmas since growth<br />
requirements vary with the species.<br />
An inoculum <strong>of</strong> 10 4 to 10 5 colour changing units (CCU) per ml has been<br />
recommended. If available, a control strain with well established MIC ranges <strong>of</strong> the<br />
tested drugs should be included to validate the test. Finally, m<strong>and</strong>atory controls such<br />
as sterile growth medium <strong>and</strong> the control strain in drug-free medium are required. The<br />
choice <strong>of</strong> test procedure is influenced by several factors such as the number <strong>of</strong> strains<br />
to be tested, their growth titer in broth or agar, <strong>and</strong> the generation time <strong>of</strong> the species.<br />
Broth dilution <strong>and</strong> especially microbroth dilution techniques have been<br />
modified for mycoplasmas <strong>and</strong> are the most widely used. The method employs<br />
mycoplasmal media with increasing antibiotic concentrations, inoculated with a<br />
st<strong>and</strong>ardized number <strong>of</strong> micro-organisms in 96-well microtiter plates. Commercially<br />
available 96-well microtiter plates precoated with antimicrobials can be ordered <strong>and</strong><br />
used with good results (Tanner <strong>and</strong> Wu, 1992; Tanner et al., 1993). Mycoplasma<br />
growth results in degradation <strong>of</strong> the metabolizable substrate such as glucose, arginin<br />
or urea, present in the medium with the resulting pH change visible as a colour change<br />
<strong>of</strong> a pH indicator. The MIC is defined as the lowest concentration <strong>of</strong> an antimicrobial<br />
agent that prevents a colour change at the time when the colour in the control without<br />
antibiotics has changed. The MIC <strong>of</strong> an antimicrobial agent for M. hyopneumoniae<br />
will only be available after 14 days <strong>of</strong> incubation. Turbidity or colour change in broth<br />
control indicates bacterial contamination.<br />
MICs <strong>of</strong> mycoplasmas growing more easily on solid agar, e.g. M. bovis<br />
(Francoz et al., 2005) <strong>and</strong> M. hominis (Waites et al., 1999), can also be determined<br />
using the E-test (AB BIODISK, Solna, Sweden). This test is based on the transfer <strong>of</strong> a<br />
continuous concentration gradient <strong>of</strong> an antimicrobial agent from a plastic strip into<br />
agar medium (Waites et al., 1997).<br />
No specific breakpoints for mycoplasmas are available, so it is preferable to<br />
merely report MICs. Some laboratories have adopted the breakpoints used for the<br />
interpretation <strong>of</strong> MICs for other bacteria (Bébéar <strong>and</strong> Bébéar, 2002). The<br />
development <strong>of</strong> antimicrobial resistance can be determined using the microbiological<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
criterion. When a monomodal Gaussian distribution is found, no acquired resistance is<br />
present, but when the distribution is bimodal or multimodal a resistance mechanism<br />
can be supposed. Also when tailing, i.e. the loss <strong>of</strong> the normal Gaussian distribution,<br />
is seen, a resistance mechanism can be supposed (Butaye, 2000).<br />
IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE TO ANTIMICROBIALS USED<br />
IN PIG VETERINARY MEDICINE<br />
Few studies are available that document the in vitro susceptibilities <strong>of</strong> M.<br />
hyopneumoniae against antimicrobials used in swine practice to treat respiratory<br />
disorders. Since M. hyopneumoniae is a fastidious organism to isolate <strong>and</strong> the<br />
procedure is time consuming, only limited numbers <strong>of</strong> isolates were used for MIC<br />
determination. Broth is preferable <strong>and</strong> most frequently used (Table 4).<br />
Inconsistencies in results were seen in the susceptibility testing <strong>of</strong><br />
tetracyclines. Williams (1978) found chlortetracycline to be less active than<br />
oxytetracycline <strong>and</strong> doxycycline, Etheridge et al. (1979) found a wide variation in the<br />
MICs (1.6 – 25 µg/ml) for chlortetracycline <strong>and</strong> Yamamoto <strong>and</strong> Koshimizu (1984)<br />
reported high MICs (≥ 40 µg/ml) for chlortetracycline. Inamoto et al. (1994) observed<br />
an increase in resistance to both chlortetracycline <strong>and</strong> oxytetracycline occurring in<br />
Japanese M. hyopneumoniae field strains isolated between 1970 <strong>and</strong> 1990. MICs <strong>of</strong><br />
unstable antimicrobials like chlortetracycline <strong>and</strong> valnemulin are unreliable since<br />
MIC determination <strong>of</strong> M. hyopneumoniae can take up to 14 days.<br />
In contrast to most studies reporting the lack <strong>of</strong> acquired resistance in M.<br />
hyopneumoniae, Wu et al. (1997) found several resistant isolates. Of the 14 isolates<br />
tested, 13 were resistant against tylosin, although 10 were susceptible to tilmicosin.<br />
One isolate was resistant to lincomycin, apramycin <strong>and</strong> enr<strong>of</strong>loxacin, two against<br />
spectinomycin. All isolates were susceptible to tetracycline <strong>and</strong> gentamicin. Because<br />
<strong>of</strong> inconsistency in the resistance pr<strong>of</strong>ile against macrolides <strong>and</strong> the fact that the M.<br />
hyopneumoniae isolates were incubated for only 3 days, the results should be<br />
interpreted with caution <strong>and</strong> are therefore not included in Table 2.<br />
In conclusion, despite some reports on resistance against tetracyclines,<br />
acquired resistance in M. hyopneumoniae does not seem to constitute a major problem<br />
to date. Genotypical confirmation <strong>of</strong> resistance has not been reported for M.<br />
hyopneumoniae.<br />
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ANTIMICROBIAL RESISTANCE IN MYCOPLASMA SPP.<br />
Intrinsic, innate or natural resistance is defined as the relative insensitivity <strong>of</strong><br />
all members <strong>of</strong> a bacterial species or genus against an antimicrobial agent. Some<br />
intrinsic resistances can be related to only one Mycoplasma species, while others can<br />
be related to all members <strong>of</strong> the class <strong>of</strong> Mollicutes. By contrast, resistance may be<br />
acquired by some strains within a species usually susceptible to the antimicrobial<br />
agent under consideration.<br />
Similar biochemical mechanisms may be responsible for intrinsic <strong>and</strong> acquired<br />
resistances, e.g. target modification, decreased uptake or enzymatic modification <strong>of</strong><br />
the antimicrobial agent.<br />
Intrinsic resistance related to the class Mollicutes<br />
Mollicutes lack a cell wall <strong>and</strong> are therefore resistant to all β-lactam<br />
antibiotics, like penicillin, ampicillin, amoxicillin, methicillin <strong>and</strong> cephalosporins.<br />
Also other antimicrobials targeting the cell wall, like glycopeptides (vancomycin,<br />
teicoplanin <strong>and</strong> avoparcin) are <strong>of</strong> no use in the treatment <strong>of</strong> mycoplasma infections. In<br />
addition, mycoplasmas are also resistant to polymyxins, sulfonamides, trimethoprim,<br />
naladixic acid <strong>and</strong> rifampin.<br />
Intrinsic resistance related to the Mycoplasma species<br />
Innate resistance related to specific Mycoplasma species concerns mainly the<br />
macrolides. As described above, macrolides are classified into three subgroups<br />
according to their lactone ring sizes (14-, 15- <strong>and</strong> 16-membered lactone ring). M.<br />
hyopneumoniae, M. flocculare, M. bovis, M. pulmonis <strong>and</strong> M. fermentans are<br />
insensitive to the 14-membered lactone ring macrolides, erythromycin <strong>and</strong><br />
ole<strong>and</strong>omycin (Williams, 1978; Tanner et al., 1993; Chambaud et al., 2001; Bébéar<br />
<strong>and</strong> Bébéar, 2002; Francoz et al., 2005), whereas M. pneumoniae <strong>and</strong> M. genitalium<br />
are susceptible (Bébéar et al., 2000; Kenny <strong>and</strong> Cartwright, 2001). The genetic basis<br />
<strong>of</strong> this innate resistance has been elucidated. Macrolides bind to the bacterial<br />
ribosome <strong>and</strong> affect its peptidyltransferase activity in the V domain <strong>of</strong> 23S rRNA. In<br />
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erythromycin <strong>and</strong> ole<strong>and</strong>omycin resistant mycoplasmas, a G →A transition at position<br />
2057 is found in the sequence <strong>of</strong> the 23S rRNA.<br />
Acquired antimicrobial resistance in Mycoplasma spp.<br />
Generally speaking, there are three main mechanisms <strong>of</strong> acquired<br />
antimicrobial resistance in mycoplasmas: active efflux mechanisms, enzymatic<br />
modification <strong>of</strong> the drug or alteration in the drug target site. Mycoplasmas are<br />
characterized by high mutation frequencies due to the limited amount <strong>of</strong> genetic<br />
information dedicated to DNA repair systems (Razin et al., 1998).<br />
1. Tetracyclines<br />
Acquired resistance to tetracyclines has been documented in human<br />
mycoplasmas <strong>of</strong> the urogenital tract: M. hominis <strong>and</strong> Ureaplasma urealyticum (Kenny<br />
<strong>and</strong> Cartwright, 1994; Waites et al., 1997). In veterinary medicine, tetracycline<br />
resistance has been described in M. bovis <strong>and</strong> M. hyopneumoniae (Inamoto et al.,<br />
1994; Thomas et al., 2003). Resistance in M. hominis <strong>and</strong> Ureaplasma spp. to<br />
tetracycline has been associated with the presence <strong>of</strong> the tetM determinant (Roberts et<br />
al., 1985; Blanchard et al., 1992). Blanchard et al. (1992) reported that all <strong>of</strong> the tetMpositive<br />
M. hominis isolates were sensitive to doxycycline, indicating that tetM does<br />
not necessarily confer resistance to this antibiotic. Recent research supports the<br />
assumption that tetracycline resistance in mollicutes can also be mediated by other<br />
genes than tetM (Taraskina et al., 2002). In veterinary mycoplasmas, no resistance<br />
mechanisms have currently been described.<br />
2. Macrolides <strong>and</strong> lincosamides<br />
Acquired resistance to macrolides is not well documented in human (M.<br />
pneumoniae, M. hominis <strong>and</strong> U. urealyticum) <strong>and</strong> animal (M. bovis <strong>and</strong> M.<br />
gallisepticum) mycoplasmas, but seems to increase considerably during the last years<br />
(Samra et al., 2002; Thomas et al., 2003; Francoz et al., 2005; Morozumi et al.,<br />
2005). In vitro selected resistant Mycoplasma strains showed mutations in the domain<br />
V <strong>of</strong> the 23S rRNA. Positions 2058, 2059 (E. coli numbering) seem to be hot spots for<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
macrolide resistance. This is in agreement with the situation in other bacteria (Lucier<br />
et al., 1995; Alvarez-Elcoro <strong>and</strong> Enzler, 1999; Pereyre et al, 2004; Wu et al., 2005).<br />
In addition, position 2062 was determined as a position responsible for resistance<br />
against 16-lactone ring macrolides (Furneri et al., 2001). Recently, mutations in the<br />
ribosomal proteins L4 <strong>and</strong> L22 have been associated with increased MIC-values in<br />
Gram-negative bacteria <strong>and</strong> Streptococcus spp. (Franceschi et al., 2004; Roberts,<br />
2004; Prunier et al., 2005). Similar mutations were found in M. pneumoniae after in<br />
vitro selection (Pereyre et al., 2004) but not in clinical isolates. The resistance<br />
mechanism in veterinary important mycoplasmas has not yet been elucidated but it is<br />
probably similar to that in human mycoplasmas.<br />
3. Pleuromutilins<br />
Development <strong>of</strong> resistance against pleuromutilins in animal-associated<br />
mycoplasmas isolated from clinical specimens has not yet been described. One<br />
publication (Gautier-Bouchardon et al., 2002) reports the development <strong>of</strong> resistance in<br />
some M. iowae isolates after repeated passages in vitro in the presence <strong>of</strong> tiamulin,<br />
but the mechanism was not described.<br />
4. Fluoroquinolones<br />
Two fluoroquinolone resistance mechanisms have been described: 1) mutation<br />
in the four target genes, gyrA, gyrB, parC <strong>and</strong> parE <strong>and</strong> 2) reduction in the level <strong>of</strong><br />
quinolone accumulation inside the cells either by efflux or lack <strong>of</strong> penetration<br />
(Hooper, 1999).<br />
In human medicine, acquired resistance has only been reported for genital<br />
mycoplasmas. The veterinary important mycoplasmas M. gallisepticum (Reinhardt et<br />
al., 2002) <strong>and</strong> M. bovis (Thomas et al., 2003) have been shown to be able to develop<br />
resistance against fluoroquinolones in vivo. Resistance is based on stepwise alterations<br />
in the four target genes but as in other Gram-positive bacteria, mainly gyrA <strong>and</strong> parC<br />
are involved (Chen <strong>and</strong> Lo, 2003). The existence <strong>of</strong> efflux mechanisms increasing the<br />
MIC has only been demonstrated in M. hominis (Raherison et al., 2002).<br />
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5. Florfenicol<br />
No resistance against florfenicol has been described in animal-associated<br />
mycoplasmas.<br />
6. Aminoglycosides <strong>and</strong> aminocyclitols<br />
Acquired resistance to aminoglycosides has been described for M. bovis<br />
(Thomas et al., 2003) <strong>and</strong> M. hyorhinis (Idowu et al., 2003). Resistance mechanisms<br />
in other bacteria include mutations in the ribosome target, presence <strong>of</strong> efflux<br />
mechanisms, or the presence <strong>of</strong> aminoglycoside-modifying enzymes (aminoglycoside<br />
phosphotransferases, aminoglycoside nucleotidyltransferases <strong>and</strong> aminoglycoside<br />
acetyltransferases) (Kotra et al., 2000). Stepwise selections <strong>of</strong> Mycoplasma strains<br />
grown in the presence <strong>of</strong> aminoglycosides or aminocyclitols result in resistance<br />
against aminoglycosides, but the resistance mechanism has not been elucidated<br />
(Hannan, 1995).<br />
PREVENTIVE MEDICATION VERSUS VACCINATION<br />
The use <strong>and</strong> efficacy <strong>of</strong> either vaccination or preventive (strategic) medication<br />
is frequently discussed among swine veterinarians <strong>and</strong> both strategies have<br />
advantages <strong>and</strong> disadvantages. Advantages <strong>of</strong> antimicrobial prevention strategies are<br />
the flexibility, antimicrobials are <strong>of</strong>ten effective against several (respiratory)<br />
pathogens <strong>and</strong> the administration is less labour-intensive since group treatments are<br />
mainly accomplished by in-feed or in-water medication. Vaccination on the other<br />
h<strong>and</strong>, does not select for antimicrobial resistance in pathogenic bacteria <strong>and</strong> in<br />
bacteria belonging to the microbiota <strong>of</strong> the animal. It also avoids risks for<br />
antimicrobial residues in slaughter pigs. While for antimicrobial treatment, an<br />
immediate effect at herd level can be expected, the effect <strong>of</strong> vaccination <strong>of</strong> young<br />
piglets will only be evident if it is continued during approximately 6 months. It is not<br />
warranted to interrupt vaccination during periods <strong>of</strong> low risk for disease outbreaks or<br />
during periods <strong>of</strong> poor market conditions (Maes et al., 2003). Vaccination against M.<br />
hyopneumoniae has as additional effect that secondary bacterial infections<br />
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(Pasteurella multocida, Actinobacillus pleuropneumoniae) less frequently occur<br />
under field conditions (Kobisch et al., 1993; Sørensen et al., 1997).<br />
Neither vaccination nor preventive medication can prevent infection <strong>and</strong><br />
adherence <strong>of</strong> M. hyopneumoniae to the ciliated cells <strong>of</strong> the respiratory tract <strong>of</strong> treated<br />
or vaccinated pigs has been demonstrated in several studies (Hannan et al., 1982;<br />
Kubo et al., 1990; Le Gr<strong>and</strong> <strong>and</strong> Kobisch 1996, Schatzmann et al., 1996, Mateusen et<br />
al., 2002).<br />
ERADICATION OF M. HYOPNEUMONIAE<br />
Eradication <strong>of</strong> enzootic pneumonia would result in serious savings each year<br />
<strong>and</strong> in improvement <strong>of</strong> the health <strong>and</strong> welfare <strong>of</strong> the pigs.<br />
A depopulation <strong>and</strong> restocking programme has been proposed <strong>and</strong> conducted<br />
in 1960 by the Pig <strong>Health</strong> Control Organisation (Goodwin <strong>and</strong> Wittlestone, 1960) <strong>and</strong><br />
eradication <strong>of</strong> herds in the UK was described by Goodwin <strong>and</strong> Wittlestone (1967).<br />
This programme was expensive <strong>and</strong> reinfections occurred in 20% <strong>of</strong> the herds.<br />
Now, eradication <strong>of</strong> M. hyopneumoniae is mainly applied in Denmark, Finl<strong>and</strong><br />
(Heinonen et al., 1999) <strong>and</strong> Switzerl<strong>and</strong> (Zimmerman et al., 1989; Zimmerman,<br />
1990). In Switzerl<strong>and</strong>, a total <strong>and</strong> partial sanitation programme was set up<br />
(Zimmermann et al., 1989). The total sanitation programme involved complete<br />
emptying <strong>of</strong> the animal facilities by selling or culling all animals. The partial<br />
sanitation programme included a piglet <strong>and</strong> gilt free (< 10 months) interval <strong>and</strong> the<br />
temporary feeding <strong>of</strong> a medicated diet during 10-14 days. The feed contained either<br />
tiamulin (6 mg/kg body weight (BW)) or a combination <strong>of</strong> chlortetracycline (20<br />
mg/kg BW), tylosin (4 mg/kg BW) <strong>and</strong> sulfadimidin (30 mg/kg BW).<br />
Another method used mainly in the US is ‘medicated early weaning (MEW)’<br />
wherein intensive medication <strong>of</strong> the sow during late gestation <strong>and</strong> immediately<br />
following parturition <strong>and</strong> <strong>of</strong> the newborn piglets is used to derive pigs free <strong>of</strong> M.<br />
hyopneumoniae. Piglets are weaned at 5 days <strong>of</strong> age, placed in an isolated nursery on<br />
a separate site <strong>and</strong> medicated for the first 10 days after weaning (Alex<strong>and</strong>er et al.,<br />
1980). Modifications <strong>of</strong> the MEW programme, based on postponing weaning until 21-<br />
25 days <strong>of</strong> age, keeping sows <strong>and</strong> nursery pigs at the same site, vaccination strategies<br />
<strong>and</strong>/or combined with medication <strong>of</strong> the sows <strong>and</strong> medications <strong>of</strong> the piglets before<br />
<strong>and</strong> after weaning were described <strong>and</strong> evaluated by Clark et al. (1994). It was<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
concluded that isolating the pigs was as effective as medication <strong>and</strong> vaccination<br />
protocols in controlling the transmission <strong>of</strong> the pathogens investigated, including M.<br />
hyopneumoniae, Bordetella bronchiseptica <strong>and</strong> Pasteurella multocida. M.<br />
hyopneumoniae was not detected when any MEW procedure was used.<br />
Antimicrobials used for eradication programmes are preferably<br />
mycoplasmacidal, like the newer fluoroquinolones, cipr<strong>of</strong>loxacin <strong>and</strong> enr<strong>of</strong>loxacin<br />
(Hannan et al., 1989) <strong>and</strong> aminoglycosides. Tiamulin is most frequently utilized to<br />
sustain eradication programmes (Mészáros et al., 1985).<br />
Although several attempts have been made to eradicate M. hyopneumoniae<br />
from a herd, reinfection frequently occurs (2.6-10% per year) (Keller, 1987,<br />
Whittlestone, 1990; Hege et al., 2002). A prominent explanation for this finding is<br />
that neighbouring herds are not free <strong>of</strong> M. hyopneumoniae (Goodwin, 1985; Jorsal<br />
<strong>and</strong> Thomsen, 1988, Wallgren et al., 1993; Hege et al., 2002). M. hyopneumoniae is<br />
able to spread over a distance <strong>of</strong> 3.2 km (Goodwin, 1985). Other statistically<br />
significant risk factors for the reinfection as determined by Hege et al. (2002) were<br />
'finishing farm', 'large mixed breeding-finishing farm', <strong>and</strong> 'parking site for pig<br />
transport vehicles close to the farm'. A protective factor was the purchase <strong>of</strong> pigs from<br />
one supplier per batch. Another explanation might be the technique used to<br />
investigate the infection status <strong>of</strong> M. hyopneumoniae <strong>of</strong> purchased pigs. Mainly<br />
ELISA techniques on serum or colostrum samples are used. ELISA has proved to be a<br />
very useful technique but recently it was demonstrated that the pigs can be colonized<br />
by low numbers <strong>of</strong> M. hyopneumoniae organisms in the lung without showing<br />
seroconversion (Thacker, 2004).<br />
Based on the cited studies, it can be stated that vaccination or antimicrobial<br />
treatment can indeed reduce the number <strong>of</strong> organisms in the lungs but these measures<br />
do not guarantee the absence <strong>of</strong> M. hyopneumoniae. MEW contributes to prevent<br />
spread <strong>of</strong> M. hyopneumoniae from sow to <strong>of</strong>fspring.<br />
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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
REFERENCES<br />
1. Alex<strong>and</strong>er, T.J.L., Thornton, K., Boon, G., Lysons, R.J. & Gush, A.F. (1980). Medicated<br />
early weaning to obtain pigs free from pathogens endemic in the herd <strong>of</strong> origin. The Veterinary<br />
Record 106, 114-119.<br />
2. Alvarez-Elcoro, S. & Enzler, M.J. (1999). The macrolides: erythromycin, clarithromycin, <strong>and</strong><br />
azithromycin. Mayo Clinic Proceedings 74, 613-634.<br />
3. Aitken, I.A., Morgan, J.H., Dalziel, R., Burch, D.G.S. & Ripley, P.H. (1999). Comparitive in<br />
vitro activity <strong>of</strong> valnemulin against porcine bacterial pathogens. The Veterinary Record 144,<br />
128.<br />
4. Arai, S., Gohara, Y., Kuwano, K. & Kawashima, T. (1992). Antimycoplasmal activities <strong>of</strong><br />
new quinolones, tetracyclines, <strong>and</strong> macrolides against Mycoplasma pneumoniae. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 36, 1322-1324.<br />
5. Bébéar, C.M. & Bébéar, C. (2002). Antimycoplasmal agents. In: Molecular biology <strong>and</strong><br />
pathogenicity <strong>of</strong> mycoplasmas. Razin, S., Herrmann, R., Editors. Kluwer Academic / Plenum<br />
Publishers, New York., pp. 545-566.<br />
6. Bébéar, C.M., Renaudin, H., Bryskier, A. & Bébéar, C. (2000). Comparitive activities <strong>of</strong><br />
telithromycin, lev<strong>of</strong>loxacin, <strong>and</strong> other antimicrobials against human mycoplasmas.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 44, 1980-1982.<br />
7. Blanchard, A., Crabb, D.M., Dybvig, K., Duffy, L.B. & Cassell, G.H. (1992). Rapid<br />
detection <strong>of</strong> tetM in Mycoplasma hominis <strong>and</strong> Ureaplasma urealyticum by PCR: tetM confers<br />
resistance to tetracycline but not necessarily to doxycyline. FEMS Microbiology Letters 74, 277-<br />
281.<br />
8. Binder, S., Le, N.B, Berner, H. & Bauer, J. (1993). The effectiveness <strong>of</strong> tilmicosin in<br />
respiratory diseases <strong>of</strong> swine. Berliner <strong>and</strong> Münchener Tierärztliche Wochenschrift 106, 6-9.<br />
9. Bousquet, E., Morvan, H., Aitken, I. & Morgan, J.H. (1997). Comparative in vitro activity <strong>of</strong><br />
doxycycline <strong>and</strong> oxytetracycline against porcine respiratory pathogens. The Veterinary Record<br />
141, 37-40.<br />
10. Bousquet, E., Pommier, P., Wessel-Robert, S., Morvan, H., Benoit-Valierque, H. & Laval,<br />
A. (1998). Efficacy <strong>of</strong> doxycycline in feed for the control <strong>of</strong> pneumonia caused by Pasteurella<br />
multocida <strong>and</strong> Mycoplasma hyopneumoniae in fattening pigs. The Veterinary Record 143, 269-<br />
272.<br />
11. Burch, D. (1984). Tiamulin feed premix in the improvement <strong>of</strong> growth performance <strong>of</strong> pigs in<br />
herds severely affected with enzootic pneumonia. The Veterinary Record 114, 209-211.<br />
12. Burch, D. (1990). Pulse dosing versus pneumonia. Pig International. 20-24.<br />
13. Burch, D. (2001). Oral antimicrobial medication pharmacokinetics in pigs. (Online)<br />
http://www.octagon-services.co.uk/articles/pharmacokinetics.htm.<br />
14. Burch, D., Jones, G., Heard, T. & Tuck, R. (1986). The synergistic activity <strong>of</strong> tiamulin <strong>and</strong> infeed<br />
treatment <strong>of</strong> bacterially complicated enzootic pneumonia in fattening pigs. The Veterinary<br />
Record 119, 108-112.<br />
15. Butaye, P. (2000). Antimicrobial resistance in Enterococcus faecium <strong>and</strong> Enterococcus faecalis<br />
strains from animals <strong>and</strong> animal-derived foods. PhD Thesis / Ghent University / Belgium.<br />
- 50 -
1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
16. Chambaud, I., Heilig, R., Ferris, S., Barbe, V., Samson, D., Galisson, F., Moszer, I.,<br />
Dybvig, K., Wroblewski, H., Viari, A., Rocha, E.P. & Blanchard, A. (2001). The complete<br />
genome sequence <strong>of</strong> the murine respiratory pathogen Mycoplasma pulmonis. Nucleic Acids<br />
Research 29, 2145-2153.<br />
17. Chauvin, C., Beloeil, P.-A., Or<strong>and</strong>, J.-P., S<strong>and</strong>ers, P. & Madec, F. (2002). A survey <strong>of</strong><br />
group-level antibiotic prescriptions in pig production in France. Preventive Veterinary Medicine<br />
55, 109-120.<br />
18. Chen, F.J. & Lo, H.J. (2003). Molecular mechanisms <strong>of</strong> fluoroquinolone resistance. Journal <strong>of</strong><br />
Microbiology, Immunology <strong>and</strong> Infection 36, 1-9.<br />
19. Chopra, I. & Roberts, M. (2001). Tetracycline antibiotics: mode <strong>of</strong> action, applications,<br />
molecular biology, <strong>and</strong> epidemiology <strong>of</strong> bacterial resistance. Microbiology <strong>and</strong> molecular<br />
biology re<strong>view</strong>s 65, 232-260.<br />
20. Clark, L.K., Hill, M.A., Kniffen, T.S., VanAlstine, W., Stevenson, G., Meyer, K.B., Wu,<br />
C.C., Scheidt, A.B., Knox, K. & Albregts, S. (1994). An evaluation <strong>of</strong> the components <strong>of</strong><br />
medicated early weaning. Swine <strong>Health</strong> <strong>and</strong> Production 2, 5-11.<br />
21. Cundliffe, E. (1990). Recognition sites for antibiotics within rRNA. In: The ribosome: structure,<br />
functions, <strong>and</strong> evolution. Hill, W.E., Dahlberg, A., Garrett, R.A., M.P.B., Schlessinger, D.,<br />
Waener, J.R.: Editors. American Society <strong>of</strong> Microbiology, Washington D.C., pp. 479-490.<br />
22. Etheridge, J., Lloyd, L. & Cottew, G. (1979). Resistance <strong>of</strong> Mycoplasma hyopneumoniae to<br />
chlortetracycline. Australian Veterinary Journal 55, 40.<br />
23. Franceschi, F., Kanyo, Z., Sherer, E.C. & Sutcliffe, J. (2004). Macrolide resistance from the<br />
ribosome perspective. Current Drug Targets - Infectious Disorders 4, 177-191.<br />
24. Francoz, D., Fortin, M., Fecteau, G. & Messier, S. (2005). Determination <strong>of</strong> Mycoplasma<br />
bovis susceptibilities against six antimicrobial agents using the E test method. Veterinary<br />
Microbiology 105, 57-64.<br />
25. Frank, H. (1989). Zur Therapie und Prophylaxe der Enzootischen Pneumonie des Schweines<br />
mit Baytril ® . Wiener Tierarztliche Monatschrift 76, 312.<br />
26. Friendship, R.M. (2000). Antimicrobial drug use in swine. In: Antimicrobial Therapy in<br />
Veterinary Medicine. J.F. Prescott, J.D. Baggot, R.D. Walker, Editors. Iowa State University<br />
Press / Ames, Third Edition, pp. 603-616.<br />
27. Friis, N.F. & Szancer, J. (1994). Sensitivity <strong>of</strong> certain porcine <strong>and</strong> bovine mycoplasmas to<br />
antimicrobial agents in a liquid medium test compared to a disc assay. Acta Veterinaria<br />
Sc<strong>and</strong>inavia 35, 389-394.<br />
28. Furneri, P.M. Rappazzo, G., Musumarra, M.P. Di Pietro, P., Catania, L.S. & Roccasalva,<br />
L.S. (2001). Two new point mutations at A2062 associated with resistance to 16-membered<br />
macrolide antibiotics in mutant strains <strong>of</strong> Mycoplasma hominis. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 45, 2958-2960.<br />
29. Ganter, M., Dudziak, D. & Delbeck, F. (1995). Treatment <strong>of</strong> swine with chronic pneumonia<br />
with chlortetracycline-medicated feed. Deutsche Tierärztliche Wochenschrift 102, 44-49.<br />
30. Gautier-Bouchardon, A.V., Reinhardt, A.K., Kobisch, M. & Kempf, I. (2002). In vitro<br />
development <strong>of</strong> resistance to enr<strong>of</strong>loxacin, erythromycin, tylosin, tiamulin <strong>and</strong> oxytetracycline<br />
in Mycoplasma gallisepticum, Mycoplasma iowae <strong>and</strong> Mycoplasma synoviae. Veterinary<br />
Microbiology 88, 47-58.<br />
31. Giroux D., Sirois G. & Martineau G.P. (1995). Gentamicin pharmacokinetics in newborn <strong>and</strong><br />
42-day-old male piglets. Journal <strong>of</strong> Veterinary Pharmacology <strong>and</strong> Therapeutics 18, 407-412.<br />
- 51 -
1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
32. Goldstein, I., Wallet, F., Robert, J., Becquemin, H., Marquette, C-H. & Rouby, J-J. (2002).<br />
Lung tissue concentrations <strong>of</strong> nebulized amikacin during mechanical ventilation in piglets with<br />
healthy lungs. American Journal <strong>of</strong> Respiratory <strong>and</strong> Critical Care Medicine 165, 171-175.<br />
33. Goodwin, R.F.W. (1979). Activity <strong>of</strong> tiamulin against Mycoplasma suipneumoniae <strong>and</strong><br />
enzootic pneumoniae <strong>of</strong> pigs. The Veterinary Record 104, 194-195.<br />
34. Goodwin, R.F.W. (1985). Apparent reinfection <strong>of</strong> enzootic-pneumonia-free herds: search for<br />
possible causes. The Veterinary Record 116, 690-694.<br />
35. Goodwin, R.F.W. & Whittlestone, P. (1960). The Veterinary Record 72, 1029.<br />
36. Goodwin, R.F.W. & Whittlestone, P. (1967). The detection <strong>of</strong> enzootic pneumonia in pig<br />
herds. I. Eight years general experience with a pilot control scheme. The Veterinary Record 81,<br />
643-647.<br />
37. Haesebrouck, F., Pasmans, F., Chiers, K., Maes, D., Ducatelle, R. & Decostere, A. (2004).<br />
Efficacy <strong>of</strong> vaccines against bacterial diseases in swine: what can we expect Veterinary<br />
Microbiology 100, 255-268.<br />
38. Hannan, P.C.T. (1995). Antibiotic susceptibility <strong>of</strong> Mycoplasma fermentans strains from<br />
various sources <strong>and</strong> the development <strong>of</strong> resistance to aminoglycosides in vitro. Journal <strong>of</strong><br />
Medical Microbiology 42, 421-428.<br />
39. Hannan, P.C.T. (2000). Guidelines <strong>and</strong> recommendations for antimicrobial minimum inhibitory<br />
concentration testing against veterinary mycoplasma species. Veterinary Research 31, 373-395.<br />
40. Hannan, P.C.T., Bhogal, B. & Fish, J. (1982). Tylosin tartrate <strong>and</strong> tiamulin effects on<br />
experimental piglet pneumonia induced with pneumonic pig lung homogenate containing<br />
mycoplasmas, bacteria <strong>and</strong> viruses. Research in Veterinary Science 33, 76-88.<br />
41. Hannan, P.C.T. & Goodwin, R.F.W. (1990). Treatment <strong>of</strong> experimental enzootic pneumonia<br />
<strong>of</strong> the pig by norfloxacin or its 6-chloro analogue. Research in Veterinary Science 49, 203-210.<br />
42. Hannan, P.C.T., O’Hanlon, P.J. & Rogers, N.H. (1989). In vitro evaluation <strong>of</strong> various<br />
quinolone antibacterial agents against veterinary mycoplasmas <strong>and</strong> porcine respiratory bacterial<br />
pathogens. Research in Veterinary Science 46, 202-211.<br />
43. Hannan, P.C.T., Windsor, H.M. & Ripley, P.H. (1997a). In vitro susceptibilities <strong>of</strong> recent<br />
field isolates <strong>of</strong> Mycoplasma hyopneumoniae <strong>and</strong> Mycoplasma hyosynoviae to valnemulin<br />
(Econor ® ), tiamulin <strong>and</strong> enr<strong>of</strong>loxacin <strong>and</strong> the in vitro development <strong>of</strong> resistance to certain<br />
antimicrobial agents in Mycoplasma hyopneumoniae. Research in Veterinary Science 63, 157-<br />
160.<br />
44. Hannan, P.C.T., Windsor, G.D., de Jong, A., Schmeer, N. & Stegeman, M. (1997b).<br />
Comparative susceptibilities <strong>of</strong> various animal-pathogenic mycoplasmas to fluoroquinolones.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 41, 2037-2040.<br />
45. Hege, R., Zimmermann, W., Scheidegger, R. & Stärk, K.D. (2002). Incidence <strong>of</strong> reinfections<br />
with Mycoplasma hyopneumoniae <strong>and</strong> Actinobacillus pleuropneumoniae in pig farms located in<br />
respiratory-disease-free regions <strong>of</strong> Switzerl<strong>and</strong> - identification <strong>and</strong> quantification <strong>of</strong> risk factors.<br />
Acta Veterinaria Sc<strong>and</strong>inavia 43, 145-156.<br />
46. Heinonen, M., Autio, T., Saloniemi, H. & Tuovinen, V. (1999). Eradication <strong>of</strong> Mycoplasma<br />
hyopneumoniae from infected swine herds joining the LSO 2000 health class. Acta Veterinaria<br />
Sc<strong>and</strong>inavia 40, 241-252.<br />
47. Henry, S.C. & Apley, M. (1999). Therapeutics. In: Straw, B.E., D’Allaire, S., Mengeling,<br />
W.L., Taylor, D.J. (Editors), Diseases <strong>of</strong> Swine, 8 th edition, Iowa State University Press, Ames,<br />
pp. 1155-1162.<br />
- 52 -
1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
48. Hooper, D.C. (1999). Mechanisms <strong>of</strong> fluoroquinolone resistance. Drug Resistance Updates 2,<br />
38-55.<br />
49. Hooper, D.C. (2002). Fluoroquinolone resistance among Gram-positive cocci. The Lancet<br />
Infectious Diseases 2, 530-538.<br />
50. Ice, A.D., Grant, A.L., Clark, L.K., Cline, T.R., Einstein, M.E., Martin, T.G. & Diekman,<br />
M.A. (1999). <strong>Health</strong> <strong>and</strong> growth performance <strong>of</strong> barrows reared in all-in/all-out or continuous<br />
flow facilities with or without a chlortetracycline feed additive. American Journal <strong>of</strong> Veterinary<br />
Research 60, 603-608.<br />
51. Idowu, A.D., Celones, M.C., Salgado, A.E., Marquez, K.M. & Pleibel, N. (2003).<br />
Characterization <strong>of</strong> gentamicin-resistant Mycoplasma hyorhinis. Biologicals 31, 175-179.<br />
52. Inamoto, T., Takahashi, H., Yamamoto, K., Nakai, Y. & Ogimoto, K. (1994). Antibiotic<br />
susceptibility <strong>of</strong> Mycoplasma hyopneumoniae isolated from swine. The Journal <strong>of</strong> Veterinary<br />
Medical Science 56, 393-394.<br />
53. Inui, T., Taira, T., Matsushita, T. & Endo, T. (1998). Pharmacokinetic properties <strong>and</strong> oral<br />
bioavailabilities <strong>of</strong> difloxacin in pig <strong>and</strong> chicken. Xenobiotica 28, 887-893.<br />
54. Jensen, C.S., Ersboll, A.K. & Nielsen, J.P. (2002). A meta-analysis comparing the effect <strong>of</strong><br />
vaccines against Mycoplasma hyopneumoniae on daily weight gain in pigs. Preventive<br />
Veterinary Medicine 54, 265-278.<br />
55. Jorsal, S. & Thomsen, B. (1988). A cox regression analysis <strong>of</strong> risk factors related to<br />
Mycoplasma suipneumoniae reinfection in Danish SPF-herds. Acta Veterinaria Sc<strong>and</strong>inavia<br />
(Suppl.) 84, 436-438.<br />
56. Jouglar, J., Borne, P., Sionneau, G., Bardon, T. & Eclache, D. (1993). Evaluation de l’intérêt<br />
de l’association tiamuline-oxytétracycline dans la prévention des bronchopneumonies du porc<br />
charcutier. Revue de Médécine Vétérinaire 144, 981-988.<br />
57. Kavanagh, N. (1994). The effect <strong>of</strong> pulse medication with a combination <strong>of</strong> tiamulin <strong>and</strong><br />
oxytetracycline on the performance <strong>of</strong> fattening pigs in a herd infected with enzootic pneumonia.<br />
Irish Veterinary Journal 47, 58-61.<br />
58. Keller, K. (1987). Swiss scheme for higher health. Pig International, June, 14-18.<br />
59. Kenny, G.E. & Cartwright, F.D. (1994). Susceptibilities <strong>of</strong> Mycoplasma hominis, Mycoplasma<br />
pneumoniae, <strong>and</strong> Ureaplasma urealyticum to new glycylcyclines in comparison with those to<br />
older tetracyclines. Antimicrobial Agents <strong>and</strong> Chemotherapy 38, 2658-2632.<br />
60. Kenny, G.E. & Cartwright, F.D. (2001). Susceptibilities <strong>of</strong> Mycoplasma hominis, Mycoplasma<br />
pneumoniae, <strong>and</strong> Ureaplasma urealyticum to GAR-936, dalfopristin, dirithromycin,<br />
evernimicin, gatifloxacin, linezolid, moxifloxacin, quinupristin-dalfopristin, <strong>and</strong> telithromycin<br />
compared to their susceptibilities to reference macrolides, tetracyclines <strong>and</strong> fluoroquinolones.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 45, 2604-2608.<br />
61. Kobisch, M., Blanchard, B. & Le Potier, M. (1993). Mycoplasma hyopneumoniae infection in<br />
pigs: duration <strong>of</strong> the disease <strong>and</strong> resistance to reinfection. Veterinary Research 24, 67-77.<br />
62. Kobisch, M. & Sibelle, C. (1982). Evaluation de l’efficacité de la tiamuline chez des porcelets<br />
infectés expérimentalement par Mycoplasma hyopneumoniae. Receuil de Médécine Vétérinaire<br />
158, 375-381.<br />
63. Kotra, L.P., Haddad, J. & Mobashery, S. (2000). Aminoglycosides: perspectives on<br />
mechanisms <strong>of</strong> action <strong>and</strong> resistance <strong>and</strong> strategies to counter resistance. Antimicrobial Agents<br />
<strong>and</strong> Chemotherapy 44, 3249-3256.<br />
- 53 -
1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
64. Kubo, M., Yamamoto, K., Ibayshi, T. & De Keyser, H. (1990). Study on the effect <strong>of</strong><br />
lincomycin against artificial infection <strong>of</strong> mycoplasmal pneumonia in swine. Proceedings 11 th<br />
IPVS congress. Lausanne, Switzerl<strong>and</strong>. 90.<br />
65. Kunesh, J. (1981). A comparison <strong>of</strong> two antibodies in treating mycoplasma pneumonia in<br />
swine. Veterinary Medicine, Small Animal Clinics 76, 871-872.<br />
66. Le Gr<strong>and</strong>, A. & Kobisch, M. (1996). Comparaison de l’utilisation d’un vaccin et d’un<br />
traitement antibiotique séquentiel dans un élevage infecté par Mycoplasma hyopneumoniae.<br />
Veterinary Research 27, 241-253.<br />
67. Léon, E.A., Madec, F., Taylor, N.M. & Kobisch, M. (2001). Seroepidemiology <strong>of</strong><br />
Mycoplasma hyopneumoniae in pigs from farrow-to-finish farms. Veterinary Microbiology 78,<br />
331-341.<br />
68. Liu, J., Fung, K.F., Chen, Z., Zeng, Z. & Zhang, J. (2003). Pharmacokinetics <strong>of</strong> florfenicol in<br />
healthy pigs <strong>and</strong> in pigs experimentally infected with Actinobacillus pleuropneumoniae.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 47, 820-823.<br />
69. Lucier, T.S., Heitzman, K., Liu, S.K. & Hu, P.C. (1995). Transition mutations in the 23S<br />
rRNA <strong>of</strong> erythromycin-resistant isolates <strong>of</strong> Mycoplasma pneumoniae. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 39, 2770-2773.<br />
70. Lukert, P. & Mulkey, G. (1982). Treatment <strong>of</strong> mycoplasmosis in young swine. Modern<br />
Veterinary Practice 63, 107-110.<br />
71. Luthman, J. & Jacobsson, S.O. (1983). The availability <strong>of</strong> tetracyclines in calves. Nordisk<br />
Veterinaer Medicin 35, 292-299.<br />
72. Maes, D. (1999). Respiratory disease in slaughter pigs: epidemiological aspects <strong>and</strong> effect <strong>of</strong><br />
vaccination against Mycoplasma hyopneumoniae. PhD thesis.<br />
73. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Lein, A., Vrijens, B. & de<br />
Kruif, A. (1998). The effect <strong>of</strong> vaccination against Mycoplasma hyopneumoniae in pig herds<br />
with a continuous production system. Journal <strong>of</strong> Veterinary Medicine, Series B 45, 495-505.<br />
74. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., Verbeke, W.,<br />
Viaene, J. & de Kruif, A. (1999). Effect <strong>of</strong> vaccination against Mycoplasma hyopneumoniae in<br />
pig herds with an all-in/all-out production system. Vaccine 17, 1024-1034.<br />
75. Maes, D., Verbeke, W., Vicca, J., Verdonck, M. & de Kruif, A. (2003). Benefit to cost <strong>of</strong><br />
vaccination against Mycoplasma hyopneumoniae in pig herds under Belgian market conditions<br />
from 1996 to 2000. Livestock Production Science 83, 85-93.<br />
76. Martineau G.P. (1980). Bronchopneumonie enzootique du porc: Amélioration de l’index<br />
pulmonaire après traitement par la tiamuline. Annales de Médécine Vétérinaire 124, 281.<br />
77. Mateusen, B., Maes, D., H<strong>of</strong>lack, G., Verdonck, M. & de Kruif, A. (2001). A comparative<br />
study <strong>of</strong> the preventive use <strong>of</strong> tilmicosin phosphate (Pulmotic premix) <strong>and</strong> Mycoplasma<br />
hyopneumoniae vaccination in a pig herd with chronic respiratory disease. Journal <strong>of</strong> Veterinary<br />
Medicine, Series B 48, 733-741.<br />
78. Mateusen, B., Maes, D., Van Goubergen, M., Verdonck, M. & de Kruif, A. (2002).<br />
Effectiveness <strong>of</strong> treatment with lincomycin hydrochloride <strong>and</strong>/or vaccination against<br />
Mycoplasma hyopneumoniae for controlling chronic respiratory disease in a herd <strong>of</strong> pigs. The<br />
Veterinary Record 151, 135-140.<br />
79. Mészáros, J., Stipkovits, L., Antal, T., Szabo, I. & Veszely, P. (1985). Eradication <strong>of</strong> some<br />
infectious pig diseases by perinatal tiamulin treatment <strong>and</strong> early weaning. The Veterinary<br />
Record 116, 8-12.<br />
- 54 -
1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
80. Morozumi, M., Hasegawa, K., Kobayashi, R., Inoue, N., Iwata, S., Kuroki, H., Kawamura,<br />
N., Nakayama, E., Tajima, T., Shimizu, K. & Ubukata, K. (2005). Emergence <strong>of</strong> macrolideresistant<br />
Mycoplasma pneumoniae with a 23S rRNA gene mutation. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 49, 2302-2306.<br />
81. Pereyre, S., Guyot, C., Renaudin, H., Charron, A., Bébéar, C. & Bébéar, C.M. (2004). In<br />
vitro selection <strong>and</strong> characterization <strong>of</strong> resistance to macrolides <strong>and</strong> related antibiotics in<br />
Mycoplasma pneumoniae. Antimicrobial Agents <strong>and</strong> Chemotherapy 48, 460-465.<br />
82. Prescott, J.F. (2000a). Aminoglycosides <strong>and</strong> aminocyclitols. In: Prescott, J.F. Baggot, J.D.,<br />
Walker, R.D. (Editors), Antimicrobial Therapy in Veterinary Medicine, 3 th edition, Iowa State<br />
University Press, Ames, pp.191-228.<br />
83. Prescott, J.F. (2000b). Tetracyclines. In: Prescott, J.F. Baggot, J.D., Walker, R.D. (Editors),<br />
Antimicrobial Therapy in Veterinary Medicine, 3 th edition, Iowa State University Press, Ames,<br />
pp. 275-289.<br />
84. Prescott, J.F. (2000c) Lincosamides, Macrolides <strong>and</strong> Pleuromutilins. In: Prescott, J.F. Baggot,<br />
J.D., Walker, R.D. (Editors), Antimicrobial Therapy in Veterinary Medicine, 3 th edition, Iowa<br />
State University Press, Ames, pp. 229-262.<br />
85. Prunier, A.L., Trong, H.N., T<strong>and</strong>e, D., Segond, C. & Leclercq, R. (2005). Mutation <strong>of</strong> L4<br />
ribosomal protein conferring unusual macrolide resistance in two independent clinical isolates <strong>of</strong><br />
Staphylococcus aureus. Microbial Drug Resistance 11, 18-20.<br />
86. Raherison, S., Gonzales, P., Renaudin, H., Charron, A., Bébéar, C. & Bébéar, C.M. (2002).<br />
Evidence <strong>of</strong> active efflux in resistance to cipr<strong>of</strong>loxacin <strong>and</strong> to ethidium bromide by Mycoplasma<br />
hominis. Antimicrobial Agents <strong>and</strong> Chemotherapy 46, 672-679.<br />
87. Razin, S., Yogev, D. & Naot, Y. (1998). Molecular biology <strong>and</strong> pathogenicity <strong>of</strong> mycoplasmas.<br />
Microbiology <strong>and</strong> Molecular Biology Re<strong>view</strong>s 62, 1094-1156.<br />
88. Reinhardt, A.K., Kempf, I., Kobisch, M. & Gautier-Bouchardon, A.V. (2002).<br />
Fluoroquinolone resistance in Mycoplasma gallisepticum: DNA gyrase as primary target <strong>of</strong><br />
enr<strong>of</strong>loxacin <strong>and</strong> impact <strong>of</strong> mutations in topoisomerase on resistance level. Journal <strong>of</strong><br />
Antimicrobial Chemotherapy 50, 589-592.<br />
89. Roberts, M.C. (1992). Antibiotic resistance. In: Mycoplasmas: molecular biology <strong>and</strong><br />
pathogenesis. Manil<strong>of</strong>f, J., McElhaney, R.N., Finch, L.R. & Baseman, J.B.: Editors. American<br />
Society for Microbiology, Washington, D.C. pp. 513-524.<br />
90. Roberts, M.C. (2004). Distribution <strong>of</strong> macrolide, lincosamide, streptogramin, ketolide <strong>and</strong><br />
oxazolidinone (MLSKO) resistance genes in Gram-negative bacteria. Current Drug Targets -<br />
Infectious Disorders 4, 207-215.<br />
91. Roberts, M.C., Koutsky, L.A., Holmes, K.K., LeBlanc, D.J. & Kenny, G.E. (1985).<br />
Tetracycline-resistant Mycoplasma hominis strains contain streptococcal tetM sequences.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 28, 141-143.<br />
92. Ross, R. & Cox, D. (1988). Evaluation <strong>of</strong> tiamulin for treatment <strong>of</strong> mycoplasmal pneumonia in<br />
swine. Journal <strong>of</strong> the American Veterinary Medical Association 193, 441-446.<br />
93. Samra, Z., Rosenberg, S. & S<strong>of</strong>fer, Y. (2002). In vitro susceptibility <strong>of</strong> Mycoplasma hominis<br />
clinical isolates to tetracyclines, quinolones <strong>and</strong> macrolides. Diagnostic Microbiology <strong>and</strong><br />
Infectious Disease 44, 359-361.<br />
94. Saux, M.C., Crockett, R., Fourtillan, J.B., Leng, B. & Couraud, L. (1986). Diffusion <strong>of</strong><br />
amikacin in the lungs. Pathologie - Biologie (Paris) 34, 113-117.<br />
- 55 -
1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
95. Schatzmann, E., Keller, H., Grest, P., Lorenz, D. & Burri, W. (1996). Feldversuche met<br />
einer Vakzine gegen die Enzootische Pneumonie (EP) der Schweine. Schweizer Archiv für<br />
Tierheilkunde 138, 483-489.<br />
96. Schuller, W., Neumeister, E. & Vogl, D. (1977). Zur Sanierung von mit Enzootischer<br />
Pneumonie versuchten Schweinebest<strong>and</strong>en. Wiener Tierärztliche Monatschrift 64, 156-160.<br />
97. Scorneaux, B. & Shryock, T.R. (1998). Intracellular accumulation, subcellular distribution <strong>and</strong><br />
efflux <strong>of</strong> tilmicosin in swine phagocytes. Journal <strong>of</strong> Veterinary Pharmacology <strong>and</strong> Therapeutics<br />
21, 257-268.<br />
98. Sørensen, V., Ahrens, P., Barfod, K., Feenstra, A.A., Feld, N.C., Friis, N.F., Bille-Hansen,<br />
V., Jensen, N.E. & Pedersen, M.W. (1997). Mycoplasma hyopneumoniae infection in pigs:<br />
duration <strong>of</strong> the disease <strong>and</strong> evaluation <strong>of</strong> four diagnostic assays. Veterinary Microbiology 54,<br />
23-34.<br />
99. Stipkovits, L., Laky, Z., Abonyi, T., Siugzdaite, J. & Szabo, I. (2003). Reduction <strong>of</strong> economic<br />
losses caused by mycoplasmal pneumonia <strong>of</strong> pigs by vaccination with Respisure <strong>and</strong> by<br />
Tiamulin treatment. Acta Veterinaria Hungaria 51, 259-271.<br />
100. Takahashi, K., Kuniyasu, C., Yoshida, Y. & Momotani, E. (1978). Sensitivity in vitro to<br />
macrolide antibiotics <strong>and</strong> tetracyclines <strong>of</strong> Mycoplasma hyopneumoniae isolated from porcine<br />
pneumonic lungs. National Institute <strong>of</strong> Animal <strong>Health</strong> Quarterly (Tokyo) 18, 41-42.<br />
101. Tanner, A.C., Erickson, B.Z. & Ross, R.F. (1993). Adaptation <strong>of</strong> the Sensititre ® broth<br />
microdilution technique to antimicrobial susceptibility testing <strong>of</strong> Mycoplasma hyopneumoniae.<br />
Veterinary Microbiology 36, 301-306.<br />
102. Tanner, A.C. & Wu, C.C. (1992). Adaptation <strong>of</strong> the Sensititre broth microdilution technique to<br />
antimicrobial susceptibility testing <strong>of</strong> Mycoplasma gallisepticum. Avian Diseases 36, 714-717.<br />
103. Taraskina, A.E., Savicheva, A.M., Akopian, T.A., Soroka, A.E., Momynaliev, K.T. &<br />
Govorun, V.M. (2002). Drift <strong>of</strong> tetM determinant in urogenital microbiocenosis containing<br />
mycoplasmas during treatment with a tetracycline antibiotic. Bulletin <strong>of</strong> Experimental Biology<br />
<strong>and</strong> Medicine 134, 60-63.<br />
104. Ter Laak, E.A., Pijpers, A., Noordergraaf, J.H., Schoevers, E.C. & Verheijden, J.H.M.<br />
(1991). Comparison <strong>of</strong> methods for in vitro testing <strong>of</strong> susceptibility <strong>of</strong> porcine Mycoplasma<br />
species to antimicrobial agents. Antimicrobial Agents <strong>and</strong> Chemotherapy 35, 228-233.<br />
105. Thacker, E.L., Thacker, B.J., Kuhn, M., Hawkins, P.A. & Waters, W.R. (2000). Evaluation<br />
<strong>of</strong> local <strong>and</strong> systemic immune response induced by intramuscular injection <strong>of</strong> a Mycoplasma<br />
hyopneumoniae bacterin to pigs. American Journal <strong>of</strong> Veterinary Research 61, 1384-1389.<br />
106. Thacker, E.L. (2004). Diagnosis <strong>of</strong> Mycoplasma hyopneumoniae. Animal <strong>Health</strong> Research<br />
Re<strong>view</strong>s 5, 317-320.<br />
107. Thomas, A., Nicolas, C., Dizier, I., Mainil, J. & Linden, A. (2003). Antibiotic susceptibilities<br />
<strong>of</strong> recent isolates <strong>of</strong> Mycoplasma bovis in Belgium. The Veterinary Record 153, 428-431.<br />
108. Thomas, E., Gr<strong>and</strong>emange, E., Pommier, P., Wessel-Robert, S. & Davot, J.L. (2000). Field<br />
evaluation <strong>of</strong> efficacy <strong>and</strong> tolerance <strong>of</strong> a 2% marb<strong>of</strong>loxacin injectable solution for the treatment<br />
<strong>of</strong> respiratory disease in fattening pigs. The Veterinary Quarterly 22, 131-135.<br />
109. Timmerman, T., Dewulf, J., Catry, B., Feyen, B., Opsomer, G., de Kruif, A. & Maes, D.<br />
(2005). Quantification <strong>and</strong> evaluation <strong>of</strong> antimicrobial-drug use in group treatments for fattening<br />
pigs in Belgium. Preventive Veterinary Medicine, accepted.<br />
110. Vester, B. & Douthwaite, S. (2001). Macrolide resistance conferred by base substitutions in<br />
23S rRNA. Antimicrobial Agents <strong>and</strong> Chemotherapy 45, 1-12.<br />
- 56 -
1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS<br />
111. Waites, K.B., Canupp, K.C. & Kenny, G.E. (1999). In vitro susceptibilities <strong>of</strong> Mycoplasma<br />
hominis to six fluoroquinolones as determined by E test. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 43, 2571-2573.<br />
112. Waites, K.B., Crabb, D.M., Duffy, L.B. & Cassel, G.H. (1997). Evaluation <strong>of</strong> the E test for<br />
detection <strong>of</strong> tetracycline resistance in Mycoplasma hominis. Diagnostic Microbiology <strong>and</strong><br />
Infectious Disease 27, 117-122.<br />
113. Wallgren, P., Sahl<strong>and</strong>er, P., Hassleback, G. & Heldmer, E. (1993). Control <strong>of</strong> infections with<br />
Mycoplasma hyopneumoniae in swine herds by disrupting the chain <strong>of</strong> infection, disinfection <strong>of</strong><br />
buildings <strong>and</strong> strategic medical treatment. Journal <strong>of</strong> Veterinary Medicine, Series B 40, 157-169.<br />
114. Wallmann, J., Kaspar, H. & Kroker, R. (2004). The prevalence <strong>of</strong> antimicrobial susceptibility<br />
<strong>of</strong> veterinary pathogens from cattle <strong>and</strong> pigs: national antibiotic resistance monitoring<br />
2002/2003 <strong>of</strong> the BVL. Berliner und Münchener Tierärztliche Wochenschrift 117, 480-492.<br />
115. Weisblum, B. (1998). Macrolide resistance. Drug Resistance Updates 1, 29-41.<br />
116. Whittlestone, P. (1990). Control <strong>of</strong> enzootic pneumonia infection in pigs. Zentralblatt (Suppl.)<br />
20, Gustav Fischer Verlag, Stuttgart, New York, pp. 254-259.<br />
117. Williams, P.P. (1978). In vitro susceptibility <strong>of</strong> Mycoplasma hyopneumoniae <strong>and</strong> Mycoplasma<br />
hyorhinis to fifty-one antimicrobial agents. Antimicrobial Agents <strong>and</strong> Chemotherapy 14, 210-<br />
213<br />
118. Wu, C.C., Shryock, T.R., Lin, T.L. & Veenhuizen, M.F. (1997). Testing antimicrobial<br />
susceptibility against Mycoplasma hyopneumoniae in vitro. Swine <strong>Health</strong> <strong>and</strong> Production 5,<br />
227-230<br />
119. Wu, C.M., Wu, H., Ning, Y., Wang, J., Du, X. & Shen, J. (2005). Induction <strong>of</strong> macrolide<br />
resistance in Mycoplasma gallisepticum in vitro <strong>and</strong> its resistance-related mutations within<br />
domain V <strong>of</strong> 23S rRNA. FEMS Microbiology Letters 247, 199-205.<br />
120. Yamamoto, K. & Koshimizu, K. (1984). In vitro susceptibility <strong>of</strong> Mycoplasma hyopneumoniae<br />
to antibiotics. In: Proceedings <strong>of</strong> the 8 th IPVS Congress Ghent, Belgium, 116.<br />
121. Zimmerman, W., Odermatt, W. & Tschudi, P. (1989). Enzootische Pneumonie (EP): die<br />
Teilsanierung EP-reinfizierter Schweinezuchtbetriebe als Alternative zur Totalsanierung.<br />
Schweizer Archiv für Tierheilkunde 131, 179-191.<br />
122. Zimmerman, W. (1990). Erfahrungen met der EP-Teilsanierung im Tilgungsprogramm des<br />
Schweizerischen Schweinegesundheitsdienstes (SGD). Tierärztliche Umschau 45, 556-562.<br />
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- 59 -<br />
1.2 AIMS
Mycoplasma hyopneumoniae is the primary cause <strong>of</strong> enzootic pneumonia, a<br />
disease which is responsible for major economic losses in the pig industry worldwide.<br />
In several studies it was shown that environmental factors influence the clinical<br />
course <strong>of</strong> the disease <strong>and</strong> the extent <strong>of</strong> economic losses in infected herds. Variation in<br />
the clinical course <strong>of</strong> the disease may, however, also be due to differences in virulence<br />
between different M. hyopneumoniae strains, as has been demonstrated for several<br />
other bacterial pathogens. No studies dealing with this subject are available in the<br />
literature. The first general aim <strong>of</strong> this thesis was therefore to evaluate the virulence <strong>of</strong><br />
M. hyopneumoniae field isolates.<br />
Antimicrobials are <strong>of</strong>ten used in the field to treat M. hyopneumoniae infected<br />
animals. As stipulated in the re<strong>view</strong> <strong>of</strong> the literature, very few data are available on<br />
the susceptibility <strong>of</strong> M. hyopneumoniae field isolates. This is mainly due to the<br />
fastidious nature <strong>of</strong> this micro-organism that is extremely hard to isolate <strong>and</strong> to<br />
cultivate, making it difficult to obtain field isolates <strong>and</strong> to test their antimicrobial<br />
susceptibility in vitro. A second general aim <strong>of</strong> this thesis was to determine the<br />
susceptibility <strong>of</strong> M. hyopneumoniae field isolates to different antimicrobials.<br />
The specific aims <strong>of</strong> this study were:<br />
- To compare patterns <strong>of</strong> M. hyopneumoniae infections in farrow-to-finish<br />
pig herds with diverging disease-course,<br />
- to evaluate the virulence <strong>of</strong> M. hyopneumoniae field isolates in an<br />
experimental infection model,<br />
- to determine in vitro susceptibility <strong>of</strong> M. hyopneumoniae field isolates to<br />
antimicrobial agents,<br />
- to characterize the genetic mechanisms <strong>of</strong> acquired resistance to<br />
macrolides <strong>and</strong> fluoroquinolones in M. hyopneumoniae,<br />
- to determine the efficacy <strong>of</strong> in feed medication with tylosin for treatment<br />
<strong>of</strong> an experimental M. hyopneumoniae infection.<br />
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2 EXPERIMENTAL STUDIES<br />
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2.1 VIRULENCE OF MYCOPLASMA HYOPNEUMONIAE<br />
ISOLATES<br />
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2.1.1 PATTERNS OF MYCOPLASMA HYOPNEUMONIAE<br />
INFECTIONS IN BELGIAN FARROW-TO-FINISH PIG HERDS WITH<br />
DIVERGING DISEASE-COURSE<br />
Modified from:<br />
PATTERNS OF MYCOPLASMA HYOPNEUMONIAE INFECTIONS IN BELGIAN FARROW-TO-<br />
FINISH PIG HERDS WITH DIVERGING DISEASE-COURSE<br />
J. VICCA, D. MAES, L. THERMOTE, J. PEETERS, F. HAESEBROUCK & A. DE KRUIF<br />
JOURNAL OF VETERINARY MEDICINE, SERIES B 2002, 49, 349-353<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
SUMMARY<br />
Patterns <strong>of</strong> Mycoplasma hyopneumoniae infections were investigated in 5<br />
clinically infected herds <strong>and</strong> in 5 herds subclinically infected with M. hyopneumoniae.<br />
In the clinically infected herds, housing <strong>and</strong> management conditions were good<br />
whereas these conditions were poor in the subclinically infected herds. In each herd,<br />
serumantibodies against M. hyopneumoniae were detected in pigs <strong>of</strong> different ages<br />
<strong>and</strong> nasal swabs were taken for M. hyopneumoniae detection using nested PCR<br />
(nPCR). The percentage <strong>of</strong> seropositive pigs in the clinically infected herds increased<br />
from 8% in pigs <strong>of</strong> 9 weeks to 52% in pigs <strong>of</strong> 18 weeks <strong>and</strong> seroconversion was most<br />
shown between 12 <strong>and</strong> 15 weeks. In the subclinically infected herds, the percentages<br />
increased from 2% to 24% <strong>and</strong> most <strong>of</strong> the pigs became seropositive between 15 <strong>and</strong><br />
18 weeks. The percentage <strong>of</strong> nPCR positive pigs at 6 weeks was 16% <strong>and</strong> 0% in the<br />
clinically <strong>and</strong> subclinically infected herds, respectively. The results demonstrate that<br />
the seroprevalences were higher in the clinically infected herds <strong>and</strong> that most <strong>of</strong> the<br />
pigs became infected with M. hyopneumoniae at a younger age. It can be concluded<br />
that additional factors different from housing <strong>and</strong> management, like differences<br />
among M. hyopneumoniae isolates, may determine the infection pattern <strong>of</strong> M.<br />
hyopneumoniae <strong>and</strong> the clinical course <strong>of</strong> the infection.<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
INTRODUCTION<br />
Enzootic pneumonia in pigs, caused by M. hyopneumoniae as primary agent,<br />
is a chronic respiratory disease that causes major economic losses to the pig industry.<br />
Infections with M. hyopneumoniae are very common worldwide, especially in<br />
countries with intensive production systems. Epidemiological studies showed that<br />
more than 90% <strong>of</strong> the swine farms in Belgium are infected with M. hyopneumoniae<br />
(Maes et al., 1999a; 2000). The disease is mainly characterized by chronic coughing<br />
<strong>and</strong> reduced performance in grow-finishing pigs, although many infections with M.<br />
hyopneumoniae occur without overt clinical symptoms (Ross, 1999). The control <strong>of</strong><br />
the disease can be accomplished in a number <strong>of</strong> different ways. First <strong>of</strong> all, the<br />
housing conditions <strong>of</strong> the pigs must be optimal <strong>and</strong> they must be appropriate<br />
according to the age <strong>of</strong> the pigs. Management practices that reduce the spread <strong>of</strong> M.<br />
hyopneumoniae infections e.g. optimal stocking density, no mixing <strong>of</strong> pigs, all-in/allout<br />
(AIAO) production, must be adopted. Additional control measures such as the<br />
preventive use <strong>of</strong> antimicrobials during periods <strong>of</strong> risk <strong>and</strong>/or vaccination against M.<br />
hyopneumoniae have been used in many swine farms. The latter measures are<br />
generally effective in reducing the severity <strong>and</strong> economic losses <strong>of</strong> the disease <strong>and</strong> in<br />
improving the performance <strong>of</strong> the pigs (Clark et al., 1991; Legr<strong>and</strong> <strong>and</strong> Kobisch,<br />
1996).<br />
In some farms, however, these measures, applied either individually or<br />
collectively, do not always control M. hyopneumoniae infections effectively. Farms<br />
with good housing conditions <strong>and</strong> management practices may suffer from clinical<br />
disease whereas in some infected farms with poor environmental conditions, M.<br />
hyopneumoniae infections do not cause obvious clinical disease. Similarly,<br />
medication <strong>and</strong> vaccination do not consistently provide the expected or desired level<br />
<strong>of</strong> control against M. hyopneumoniae infections (Mateusen et al., 2002). The<br />
beneficial effects <strong>of</strong> vaccination against M. hyopneumoniae vary from farm to farm<br />
(Morrow et al., 1994; Maes et al., 1999b) <strong>and</strong> the results cannot consistently be<br />
predicted beforeh<strong>and</strong>. Therefore, it can be expected that besides housing <strong>and</strong><br />
management conditions, other factors e.g. differences in M. hyopneumoniae isolates<br />
(Frey et al., 1992; Artiushin <strong>and</strong> Minion, 1996) may also determine the clinical course<br />
<strong>and</strong> infection pattern <strong>of</strong> M. hyopneumoniae.<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
The present field study aimed to investigate the infection pattern <strong>of</strong> M.<br />
hyopneumoniae in farms with either good or poor housing <strong>and</strong> management<br />
conditions. According to previous studies (Yagihashi et al., 1993; Horst et al., 1997),<br />
one should expect a higher prevalence <strong>and</strong> earlier infection <strong>of</strong> M. hyopneumoniae in<br />
the herds with poor housing <strong>and</strong> management. In addition, one should expect a higher<br />
chance for clinical disease in these farms (Pointon et al., 1985; Christensen et al.,<br />
1999). However, we intentionally selected 5 farms with poor housing <strong>and</strong><br />
management that were subclinically infected with M. hyopneumoniae, <strong>and</strong> 5 farms<br />
with good housing <strong>and</strong> management that were clinically infected with M.<br />
hyopneumoniae. The hypothesis was that, if the prevalence <strong>of</strong> M. hyopneumoniae is<br />
higher in the clinically infected herds with good housing <strong>and</strong> management conditions<br />
<strong>and</strong> if pigs are infected at a younger age in these herds, then additional factors other<br />
than housing <strong>and</strong> management also determine the course <strong>of</strong> the infection within these<br />
swine farms. The infection pattern was assessed using serology <strong>and</strong> nested PCR<br />
(nPCR) on nasal swabs <strong>of</strong> pigs <strong>of</strong> different age groups.<br />
MATERIALS AND METHODS<br />
Selection <strong>and</strong> description <strong>of</strong> the study farms<br />
Ten farrow-to-finish pig herds, located in 4 different provinces <strong>of</strong> Fl<strong>and</strong>ers<br />
(Belgium), participated in the study (Table 1). They were selected by contacting the<br />
herd health veterinarian. The investigator visited all herds once. All the herds had at<br />
least 100 sows <strong>and</strong> they were infected with M. hyopneumoniae as demonstrated by<br />
previous laboratory data. <strong>Herd</strong>s that had used M. hyopneumoniae vaccination in the<br />
past 3 years were excluded. The breeding population mainly consisted <strong>of</strong> hybrid sows<br />
that were either purchased or produced by on-farm selection. The sows were<br />
inseminated with semen from Piétrain boars for the production <strong>of</strong> slaughter pigs. The<br />
parity distribution <strong>of</strong> the sows was very similar between the selected farms. On<br />
average, sows had 3.5 to 4.5 litters before they were culled. In all the herds, pigs were<br />
weaned when they were about 4 weeks old. At that time, they were transferred to the<br />
nursery unit in which they were housed until 10-12 weeks <strong>of</strong> age. Thereafter, they<br />
were moved to the grow-finishing unit in which they remained until slaughter age (6-<br />
7 months). A fully slatted floor was present in the grow-finishing units <strong>of</strong> all farms.<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
Table 1: Some important characteristics <strong>of</strong> the 10 selected herds infected with<br />
Mycoplasma hyopneumoniae that participated in the study<br />
<strong>Herd</strong><br />
Infection<br />
Pig density in the<br />
<strong>Herd</strong> size<br />
Purchase <strong>of</strong><br />
Production system<br />
status a<br />
Municipality<br />
(number <strong>of</strong><br />
gilts<br />
(# <strong>of</strong> pigs/km²)<br />
sows)<br />
1 0 1123 120 No AIAO in N,<br />
Continuous in G-F b<br />
2 0 1363 220 Yes AIAO in N,<br />
Continuous in G-F<br />
3 0 845 120 Yes Continuous<br />
4 0 393 120 No Continuous<br />
5 0 1413 200 No Continuous<br />
6 1 1363 240 Yes AIAO<br />
7 1 1472 100 Yes AIAO<br />
8 1 1123 100 Yes AIAO<br />
9 1 1123 200 No AIAO<br />
10 1 875 300 No AIAO<br />
a<br />
b<br />
0 subclinically infected herds; 1 clinically infected herds<br />
AIAO all-in/all-out; N nursery unit; G-F grow-finishing unit<br />
In 5 <strong>of</strong> the 10 herds, the housing conditions <strong>and</strong> management practices were<br />
optimal although the herds were clinically infected with M. hyopneumoniae for at<br />
least one year. Good housing conditions implied the presence <strong>of</strong><br />
compartmentalization, indirect air-inlets so that the incoming air is warmed before<br />
reaching the pig area, <strong>and</strong> programmes to maintain appropriate temperatures in the<br />
rooms according to the age <strong>of</strong> the pigs. Compartmentalization is the subdivision <strong>of</strong> a<br />
barn into rooms with a capacity <strong>of</strong> about 100-150 pigs <strong>and</strong> with its own ventilation<br />
system. The management practices in these farms included the use <strong>of</strong> all-in/all-out<br />
(AIAO) production in the nursery <strong>and</strong> grow-finishing units, maintaining optimal<br />
stocking densities <strong>of</strong> the pigs, <strong>and</strong> practicing strict internal <strong>and</strong> external biosecurity<br />
measures. According to the swine producer, the herd health veterinarian <strong>and</strong> the<br />
investigator, the herds were all clinically infected with M. hyopneumoniae. This was<br />
evidenced by the presence <strong>of</strong> chronic coughing in nursery <strong>and</strong> grow-finishing pigs,<br />
<strong>and</strong> by reduced production parameters in grow-finishing pigs (reduced average daily<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
weight gain, heterogeneous growth, <strong>and</strong> poor feed conversion) compared to similar<br />
herds without chronic coughing. The clinical course <strong>of</strong> the disease <strong>and</strong> data from<br />
diagnostic laboratories indicated that the problems were not primarily due to<br />
infections with other respiratory pathogens such as Actinobacillus pleuropneumoniae<br />
or swine influenza viruses.<br />
In the 5 remaining herds, the housing conditions <strong>and</strong> management practices<br />
were poor although the herds were not clinically infected with M. hyopneumoniae.<br />
The swine barns were old <strong>and</strong> worn out, there was no compartmentalization, no<br />
indirect air-inlet, <strong>and</strong> no specific programmes to maintain appropriate temperatures in<br />
the rooms. A continuous production system was practised or AIAO was used only in<br />
the farrowing <strong>and</strong> nursery unit, the stocking density in grow-finishing pigs was too<br />
high (
2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
Nasal samples <strong>and</strong> nPCR for M. hyopneumoniae<br />
Nasal swabs were collected in each herd from pigs belonging to 4 different<br />
age groups namely 6, 9, 12 <strong>and</strong> 15 weeks. Five pigs per age category were selected.<br />
The pigs <strong>of</strong> the youngest age group were selected r<strong>and</strong>omly at pen-level; those <strong>of</strong> the<br />
other age groups were r<strong>and</strong>omly selected out <strong>of</strong> the pigs that were blood sampled. The<br />
pigs <strong>of</strong> 6 <strong>and</strong> 9 weeks old were housed in the nursery unit, those <strong>of</strong> 12 <strong>and</strong> 15 weeks<br />
in the grow-finishing unit. None <strong>of</strong> the sampled pigs had received antimicrobials in<br />
the last 4 weeks.<br />
The nasal swabs were 15 cm long <strong>and</strong> they were inserted into the nostrils as deeply as<br />
possible. The swabs were transported in 700 µL PBS with 0.1% Triton x-100, <strong>and</strong><br />
they were vortexed <strong>and</strong> discarded upon arrival in the laboratory. The transport<br />
medium was heated at 100°C for 5 min <strong>and</strong> stored frozen at –25°C until PCR was<br />
performed. Five µL <strong>of</strong> undiluted sample <strong>and</strong> 5 µL <strong>of</strong> 10-fold diluted sample were used<br />
in the nPCR, which was performed as described by Stärk et al. (1998). PCR products<br />
were analyzed by electrophoresis on 1% agarose gels in TBE buffer <strong>and</strong> visualized<br />
under UV illumination after staining in Ethidium Bromide.<br />
Statistical analysis<br />
The percentage <strong>of</strong> seropositive pigs <strong>and</strong> the percentage <strong>of</strong> nPCR positive pigs<br />
in each age category were compared between the clinically <strong>and</strong> the subclinically<br />
infected herds by means <strong>of</strong> Fisher's Exact Tests, performed on the raw numeric data.<br />
The average OD-values <strong>of</strong> the different age groups were compared between the<br />
clinically <strong>and</strong> the subclinically infected herds using two-sample t-tests. Variables<br />
were considered to be significant when P-values were lower than 0.05 (two-sided).<br />
Data were analyzed using the statistical package SPSS 10.0 for Windows (SPSS 10,<br />
SPSS Inc. Illinois 60611 USA 1999).<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
RESULTS<br />
Blood samples <strong>and</strong> serology for M. hyopneumoniae<br />
The percentage <strong>of</strong> seropositive pigs in the clinically infected herds increased<br />
from 8% in pigs <strong>of</strong> 9 weeks to 52% in pigs <strong>of</strong> 18 weeks, whereas in the subclinically<br />
infected herds the percentages in the same age categories increased from 2% to 24%<br />
(Table 2). At 15 <strong>and</strong> 18 weeks, the percentage <strong>of</strong> seropositive pigs was significantly<br />
higher in the clinically infected herds than in the subclinically infected herds<br />
(P
2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
nPCR positive pigs was almost similar in the clinically (8 <strong>and</strong> 12%, respectively) <strong>and</strong><br />
subclinically (12 <strong>and</strong> 12%, respectively) infected herds (P>0.05).<br />
Table 2. The percentage <strong>of</strong> seropositive pigs a <strong>and</strong> the average OD-values b in the<br />
different age categories <strong>of</strong> 10 farrow-to-finish pig herds that were either clinically (n=5)<br />
or subclinically (n=5) infected with Mycoplasma hyopneumoniae<br />
% <strong>of</strong> seropositive pigs (min.-max.) Average OD-value (SD)<br />
Age<br />
(Weeks)<br />
Clinically<br />
infected herds<br />
Subclinically<br />
infected herds<br />
P-value<br />
Clinically<br />
infected herds<br />
Subclinically<br />
infected herds<br />
P-value<br />
9 8 (0-40) 2 (0-10) 0.362 79.1 (18.27) 82.3 (21.79) 0.088<br />
12 8 (0-40) 0 (0- 0) 0.117 79.8 (16.80) 83.7 (09.02) 0.157<br />
15 20 (0-70) 2 (0-10) 0.008 69.7 (24.36) 83.5 (13.36) 0.013<br />
18 52 (0-100) 24 (0-40) 0.007 53.0 (26.71) 64.7 (22.63) 0.021<br />
a<br />
b<br />
10 pigs per age category were r<strong>and</strong>omly selected at pen-level in each herd<br />
Average OD-values (%) were based on all investigated pigs per age category<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
Table 3. The results <strong>of</strong> the nested PCR (nPCR) on nasal swabs from pigs <strong>of</strong> different age<br />
categories in 10 farrow-to-finish pig herds that were clinically (n=5) or subclinically<br />
(n=5) infected with Mycoplasma hyopneumoniae<br />
Age category (weeks)<br />
% (min.-max.) <strong>of</strong> positive pigs a, b<br />
Clinically infected herds<br />
Subclinically infected herds<br />
6 16 (0-40) 0 (0-0)<br />
9 24 (0-40) 12 (0-20)<br />
12 8 (0-20) 12 (0-40)<br />
15 12 (0-40) 12 (0-40)<br />
a<br />
b<br />
5 pigs per age category were r<strong>and</strong>omly selected in each herd<br />
there were no significant differences between both types <strong>of</strong> herds (P>0.05)<br />
DISCUSSION<br />
The study assessed the patterns <strong>of</strong> M. hyopneumoniae infections in farms with<br />
good housing <strong>and</strong> management conditions that were clinically infected with M.<br />
hyopneumoniae, <strong>and</strong> in farms with poor housing <strong>and</strong> management conditions that<br />
were subclinically infected with M. hyopneumoniae. The hypothesis was that, if the<br />
prevalence <strong>of</strong> M. hyopneumoniae is higher in the clinically infected herds <strong>and</strong> if pigs<br />
are infected at a younger age in these herds, then additional factors different from the<br />
housing <strong>and</strong> management e.g. differences among circulating M. hyopneumoniae<br />
isolates, are associated with the course <strong>of</strong> the infection within swine farms. A<br />
difference in infection pattern due to a different parity <strong>of</strong> the sows in the herds is very<br />
unlikely since all herds had a comparable parity distribution. Although it cannot be<br />
ruled out, it is also unlikely that infections with other pathogens like PRRSV or<br />
Circovirus type 2 may have accounted for the different M. hyopneumoniae infection<br />
pattern in the selected farms since almost all Belgian herds are infected with PRRSV<br />
(Maes et al., 1997) <strong>and</strong> Circovirus type2 (Labarque et al., 2001). It is generally<br />
accepted that the housing <strong>and</strong> management conditions play a key-role in the spread<br />
<strong>and</strong> clinical course <strong>of</strong> M. hyopneumoniae infections in swine farms (Clark et al.,<br />
1991; Ross, 1999). In this respect, one should have expected a more intensive spread<br />
in the herds with poor housing <strong>and</strong> management conditions. The results <strong>of</strong> the<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
serology <strong>and</strong> nPCR however, demonstrated that this was not the case. Moreover, the<br />
seroprevalence <strong>of</strong> M. hyopneumoniae was higher <strong>and</strong> most <strong>of</strong> the pigs became<br />
infected with M. hyopneumoniae at a younger age in the clinically infected herds with<br />
good housing <strong>and</strong> management than in the subclinically infected herds with poor<br />
housing <strong>and</strong> management.<br />
The results <strong>of</strong> the present study do not underestimate the importance <strong>of</strong><br />
housing <strong>and</strong> management factors in the transmission <strong>of</strong> M. hyopneumoniae infections<br />
in swine farms (Ross, 1999). The results demonstrate that the presence <strong>of</strong> poor<br />
housing <strong>and</strong> management in M. hyopneumoniae infected herds does not automatically<br />
imply that the clinical symptoms associated with M. hyopneumoniae infections will be<br />
severe, or that the spread <strong>of</strong> M. hyopneumoniae infections will be more intensive than<br />
in other farms. Conversely, the results also demonstrate that good housing <strong>and</strong><br />
management practices do not always result in a subclinical course <strong>of</strong> M.<br />
hyopneumoniae infection. It can thus be deduced that in addition to housing <strong>and</strong><br />
management, other factors are involved in the transmission <strong>of</strong> M. hyopneumoniae<br />
infections in swine farms. These factors are currently not yet fully understood, but it is<br />
reasonable to assume that differences among M. hyopneumoniae isolates concerning<br />
the antigenic, pathogenic <strong>and</strong> genetic characteristics may account for the different<br />
infection patterns in both types <strong>of</strong> farms. Some studies have shown some evidence for<br />
differences among M. hyopneumoniae isolates (Frey et al., 1992; Artiushin <strong>and</strong><br />
Minion, 1996). Further research is currently performed in our laboratory to investigate<br />
the characteristics <strong>of</strong> the M. hyopneumoniae isolates from these farms.<br />
Although specific criteria have been used to select both groups <strong>of</strong> farms, the<br />
individual farms belonging to either the clinically or subclinically infected groups<br />
were not identical. The housing <strong>and</strong> management factors that are important for the<br />
respiratory health status <strong>of</strong> the pigs are very diverse <strong>and</strong> encompass many different<br />
aspects (Christensen et al., 1999). In this respect, it is difficult to select a group <strong>of</strong><br />
farms with all exactly the same housing <strong>and</strong> management characteristics. However, in<br />
the present study, the most important factors <strong>of</strong> the housing <strong>and</strong> management that are<br />
known to be associated with enzootic pneumonia or with respiratory disease were<br />
considered for the selection <strong>of</strong> the farms. Likewise, it is hard to find farms with<br />
identical clinical symptoms <strong>of</strong> enzootic pneumonia in all age groups or in which<br />
clinical symptoms <strong>of</strong> enzootic pneumonia are totally absent. Important symptoms <strong>of</strong><br />
enzootic pneumonia namely chronic coughing <strong>and</strong> reduced performance in grow-<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
finishing pigs (Ross, 1999) have been considered for the selection procedure, <strong>and</strong><br />
other respiratory diseases have been excluded. Thus, the farms belonging to the same<br />
group in the present study were not identical with respect to housing, management<br />
<strong>and</strong> clinical symptoms <strong>of</strong> M. hyopneumoniae infection, but they were very alike<br />
concerning these factors.<br />
The infection pattern <strong>of</strong> M. hyopneumoniae in the farms was assessed using<br />
serology <strong>and</strong> nPCR on nasal swabs <strong>of</strong> pigs <strong>of</strong> different age groups. Both diagnostic<br />
techniques have been shown to be valuable tools in pr<strong>of</strong>iling herds for M.<br />
hyopneumoniae infection (Calsamiglia et al., 1999). Serological testing is easy to<br />
perform <strong>and</strong> the DAKO ® Mh ELISA has a high sensitivity <strong>and</strong> specificity to detect<br />
serum antibodies following M. hyopneumoniae infection (Sørensen et al., 1992;<br />
1997). However, the serological response following M. hyopneumoniae infection is<br />
variable <strong>and</strong> the time-span between infection <strong>and</strong> seroconversion can take 3 to 8<br />
weeks (Nicolet et al., 1990; Kobisch et al., 1993; Morris et al., 1995). In addition,<br />
there is no correlation between the titer <strong>of</strong> serum antibodies against M.<br />
hyopneumoniae <strong>and</strong> the degree <strong>of</strong> protection against infection (Murphy et al., 1993;<br />
Yagihashi et al., 1993). Testing with nPCR on nasal swabs is a direct measure for<br />
detection <strong>of</strong> presence <strong>of</strong> nucleic acid <strong>of</strong> M. hyopneumoniae organisms. Consequently,<br />
a positive result does not automatically mean that this pig is actively shedding viable<br />
M. hyopneumoniae organisms. False negative results may arise if only negligible<br />
quantities <strong>of</strong> M. hyopneumoniae organisms are present in the nose, <strong>and</strong>/or if the nasal<br />
swab contains a lot <strong>of</strong> cellular debris that can inhibit the PCR (Stärk et al., 1992).<br />
The results <strong>of</strong> the nPCR in the present study corroborate with the serological<br />
results since the number <strong>of</strong> positive pigs in both tests was higher in the clinically<br />
infected herds <strong>and</strong> since both tests demonstrated that pigs became infected at a<br />
younger age in the clinically infected herds. However, the infection can be detected<br />
earlier with nPCR than with serology because there was a higher proportion <strong>of</strong><br />
nursery pigs that was positive using nPCR testing compared to serology. This finding<br />
corroborates with results <strong>of</strong> previous studies (Calsamiglia et al., 1999) <strong>and</strong> it indicates<br />
that nPCR testing is more appropriate than serology to detect M. hyopneumoniae<br />
infection in young pigs at the farm level. In the clinically infected herds, 16% <strong>of</strong> the<br />
pigs at 6 weeks were already positive using nPCR. This means that they became<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
infected at the beginning <strong>of</strong> the nursery period or possibly during the farrowing<br />
period. It also indicates that good housing <strong>and</strong> management do not guarantee per se<br />
that the occurrence <strong>of</strong> M. hyopneumoniae infection will be delayed until the growfinishing<br />
unit. No pigs younger than 9 weeks were blood sampled to avoid possible<br />
interference with maternal antibodies. Morris et al. (1994) showed that maternal<br />
antibodies against M. hyopneumoniae can persist as long as 9 weeks in pigs derived<br />
from sows with very high titres <strong>of</strong> serum antibodies. Although it cannot be ruled out<br />
with certainty, it is unlikely that the positive ELISA titres in a few pigs <strong>of</strong> 9 weeks<br />
were due to maternal antibodies. The number <strong>of</strong> seropositive pigs gradually increased<br />
towards the end <strong>of</strong> the finishing period. This reflects the chronic nature <strong>of</strong> M.<br />
hyopneumoniae infections (Ross, 1999). Even within clinically or subclinically<br />
infected herds, individual pigs started seroconverting at different ages (data not<br />
shown). In this respect, it is difficult to assess ‘the’ age <strong>of</strong> infection in a farm. It may<br />
be more relevant to determine the age at which most <strong>of</strong> the pigs became infected.<br />
CONCLUSION<br />
Even with a limited number <strong>of</strong> samples <strong>and</strong> the combination <strong>of</strong> nPCR on nasal<br />
swabs <strong>and</strong> serology, it was possible to assess the dynamics <strong>of</strong> M. hyopneumoniae<br />
infection in the selected farms <strong>and</strong> to elucidate differences in infection patterns<br />
between the selected clinically <strong>and</strong> subclinically infected herds. More studies also<br />
including subclinically infected farms with good housing <strong>and</strong> management <strong>and</strong><br />
clinically infected farms with poor housing <strong>and</strong> management are needed to confirm<br />
the present findings. The results <strong>of</strong> this study document that additional factors<br />
different from the investigated housing <strong>and</strong> management conditions may determine<br />
the infection pattern <strong>of</strong> M. hyopneumoniae <strong>and</strong> the clinical course <strong>of</strong> the infection.<br />
Further research will focus on possible mechanisms that can explain the different<br />
infection patterns <strong>and</strong> on possible differences among M. hyopneumoniae isolates from<br />
these farms.<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
ACKNOWLEDGEMENTS<br />
This research was financially supported by grant S-5940 (section II) <strong>of</strong> the<br />
Belgian Ministry <strong>of</strong> Small Enterprises, Trader <strong>and</strong> Agriculture, Research Foundation<br />
(Brussels). The swine producers <strong>and</strong> herd health veterinarians are acknowledged for<br />
their participation in this study.<br />
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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
REFERENCES<br />
1. Artiushin, S. & Minion, C.F. (1996). Arbitrarily primed PCR analysis <strong>of</strong> Mycoplasma<br />
hyopneumoniae field isolates demonstrates genetic heterogeneity. International Journal <strong>of</strong><br />
Systematic Bacteriology 46, 324-328.<br />
2. Calsamiglia, M., Pijoan, C. & Bosch, G. (1999). Pr<strong>of</strong>iling Mycoplasma hyopneumoniae in<br />
farms using serology <strong>and</strong> a nested PCR technique. Swine <strong>Health</strong> <strong>and</strong> Production 7, 263-268.<br />
3. Clark, L.K., Scheidt, A.B., Armstrong, C.H., Knox, K. & Mayrose, V.B. (1991). The effect<br />
<strong>of</strong> all-in/all-out management on pigs from a herd with enzootic pneumonia. Veterinary Medicine<br />
86, 946-951.<br />
4. Christensen, G., Sørensen, V. & Mousing, J. (1999). Diseases <strong>of</strong> the respiratory system. In:<br />
Leman A.D., Straw B.E., Mengeling W.L., Allaire S.D., Taylor D.J (Eds.), Diseases <strong>of</strong> Swine,<br />
8th Edition, Iowa State University Press, Ames, pp. 913-940.<br />
5. Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F. & Meyling, A. (1992). A monoclonal blocking<br />
ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. Veterinary Microbiology 30,<br />
35-46.<br />
6. Frey J., Haldimann, A. & Nicolet, J. (1992). Chromosomal heterogeneity <strong>of</strong> various<br />
Mycoplasma hyopneumoniae field strains. International Journal <strong>of</strong> Systemic Bacteriology 42,<br />
275-280.<br />
7. Horst, I., Lindner, A., Krüger, M., Gindele, H. & Sting, R. (1997). Verbreitung der<br />
Mycoplasma hyopneumoniae infektion in Deutschl<strong>and</strong>. Schluβfolgerungen für die Bekämpfung<br />
der Enzootischen Pneumonie der Schweine. Tierärztliche Umschau 52, 508-514.<br />
8. Kobisch, M., Blanchard, B. & Le Potier, M.F. (1993). Mycoplasma hyopneumoniae infection<br />
in pigs: duration <strong>of</strong> the disease <strong>and</strong> resistance to reinfection. Veterinary Research 24, 67-77.<br />
9. Labarque, G.G., Nauwynck, H.J., Mesu, A.P. & Pensaert, M.B. (2001). Seroprevalence <strong>of</strong><br />
porcine circovirus types 1 <strong>and</strong> 2 in the Belgian pig population. The Veterinary Quarterly 22,<br />
234-236.<br />
10. LeGr<strong>and</strong>, A. & Kobisch, M. (1996). Comparison <strong>of</strong> the use <strong>of</strong> a vaccine <strong>and</strong> sequential<br />
antibiotic treatment in a herd infected with Mycoplasma hyopneumoniae. Veterinary Medicine<br />
27, 241-253.<br />
11. Maes, D.G., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A.<br />
(1997). Descriptive epidemiological aspects <strong>of</strong> the seroprevalence <strong>of</strong> five respiratory disease<br />
agents in slaughter pigs from fattening pig herds. In: Proc. 8 th ISVEE Congress Paris France,<br />
05.B.19.<br />
12. Maes, D.G., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A.<br />
(1999a). Risk indicators for the seroprevalence <strong>of</strong> Mycoplasma hyopneumoniae, porcine<br />
Influenza viruses <strong>and</strong> Aujeszky’s disease virus in slaughter pigs from fattening pig herds.<br />
Journal <strong>of</strong> Veterinary Medicine, Serie B 46, 341-352.<br />
13. Maes, D.G., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., Verbeke, W.<br />
& Viaene, J. (1999b). Effect <strong>of</strong> vaccination against Mycoplasma hyopneumoniae in pig herds<br />
with an all-in/all-out production system. Vaccine 17, 1024-1034.<br />
14. Maes, D.G., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A.<br />
(2000). <strong>Herd</strong> factors associated with the seroprevalences <strong>of</strong> four major respiratory pathogens in<br />
slaughter pigs from farrow-to-finish pig herds. Veterinary Research 31, 313-327.<br />
15. Mateusen, B., Maes, D., Van Goubergen, M., Verdonck, M. & de Kruif, A. (2002). The<br />
effectiveness <strong>of</strong> strategic medication with lincomycin hydrochloride <strong>and</strong>/or vaccination against<br />
- 82 -
2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS<br />
16. Mycoplasma hyopneumoniae to control chronic respiratory disease in a swine herd. The<br />
Veterinary Record 151, 135-140.<br />
17. Morris, C.R., Gardner, I.A., Hietala, S.K., Carpenter, T.E., Anderson, R.J. & Parker,<br />
K.M. (1994). Persistence <strong>of</strong> passively acquired antibodies to Mycoplasma hyopneumoniae in a<br />
swine herd. Preventive Veterinary Medicine 21, 29-41.<br />
18. Morris, C.R., Gardner, I.A., Hietala, S.K., Carpenter, T.E., Anderson, R.J. & Parker,<br />
K.M. (1995). Seroepidemiologic study <strong>of</strong> natural transmission <strong>of</strong> Mycoplasma hyopneumoniae<br />
in a swine herd. Preventive Veterinary Medicine 21, 323-337.<br />
19. Morrow, M.W.E., Iglesias, G., Stanislaw, C., Stephenson, A. & Erickson, G. (1994). Effect<br />
<strong>of</strong> a mycoplasma vaccine on average daily gain in swine. Swine <strong>Health</strong> <strong>and</strong> Production 2, 13-18.<br />
20. Murphy, D.A., Van Alstine, W.G., Clark, L.K., Albregts, S.H. & Knok, K. (1993). Aerosol<br />
vaccination <strong>of</strong> pigs against Mycoplasma hyopneumoniae. American Journal <strong>of</strong> Veterinary<br />
Research 54, 1874-1880.<br />
21. Nicolet, J., Zimmerman, W., Chastonay, M. (1990). Epidemiology <strong>and</strong> serodiagnosis <strong>of</strong><br />
Mycoplasma hyopneumoniae. Zentralblatt für Bakteriologie Supplement 20 - Gustav Fischer<br />
Verlag, Stuttgart, New York, pp. 249-253.<br />
22. Pointon, A.M., Heap, P. & McCloud, P. (1985). Enzootic pneumonia in pigs in South<br />
Australia - factors relating to incidence <strong>of</strong> disease. Australian Veterinary Journal 62, 98-100.<br />
23. Ross, R.F. (1999).: Mycoplasmal diseases. In: Leman A.D., Straw B.E., Mengeling W.L.,<br />
Allaire S.D., Taylor D.J (Eds.), Diseases <strong>of</strong> Swine, 8th Edition, Iowa State University Press,<br />
Ames, pp. 495-509.<br />
24. Sørensen, V., Ahrens, P., Barfod, K., Feenstra, A.A., Feld, N.C., Friis, N.F., Bille-Hansen,<br />
V., Jensen, N.E. & Pedersen, M.W. (1997). Mycoplasma hyopneumoniae infection in pigs:<br />
duration <strong>of</strong> the disease <strong>and</strong> evaluation <strong>of</strong> four diagnostic assays. Veterinary Microbiology 54,<br />
23-34.<br />
25. Sørensen, V., Barfod, K. & Feld, N.C. (1992). Evaluation <strong>of</strong> a monoclonal blocking ELISA<br />
<strong>and</strong> IHA for antibodies to Mycoplasma hyopneumoniae in SPF pigs. The Veterinary Record 130,<br />
488-490.<br />
26. Stärk, K.D., Nicolet, J. & Frey, J. (1998). Detection <strong>of</strong> Mycoplasma hyopneumoniae by air<br />
sampling with a nested PCR assay. Applied Environmental Microbiology 64, 543-548.<br />
27. Yagihashi, T., Kazama, S. & Tajima, M. (1993). Seroepidemiology <strong>of</strong> mycoplasmal<br />
pneumonia <strong>of</strong> swine in Japan as surveyed by an Enzyme-Linked Immunosorbent Assay.<br />
Veterinary Microbiology 34, 155-166.<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE<br />
FIELD ISOLATES<br />
Modified from:<br />
EVALUATION OF VIRULENCE OF MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES<br />
J. VICCA, T. STAKENBORG, D. MAES, P. BUTAYE, J. PEETERS, A. DE KRUIF & F. HAESEBROUCK<br />
VETERINARY MICROBIOLOGY 2003, 97, 177-190<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
SUMMARY<br />
The course <strong>of</strong> enzootic pneumonia, caused by Mycoplasma hyopneumoniae, is<br />
strongly influenced by management <strong>and</strong> housing conditions. Other factors, including<br />
differences in virulence between M. hyopneumoniae isolates, may also be involved.<br />
The aim <strong>of</strong> this study was to evaluate the virulence <strong>of</strong> six M. hyopneumoniae field<br />
isolates <strong>and</strong> link it to genetic differences as determined by r<strong>and</strong>omly-amplified<br />
polymorphic DNA (RAPD) analysis.<br />
Ninety, conventional M. hyopneumoniae-free piglets were inoculated<br />
intratracheally with the field isolates, a virulent reference strain or sterile culture<br />
medium. Animals were examined daily for the presence <strong>of</strong> disease signs <strong>and</strong> a<br />
respiratory disease score (RDS) was assessed per pig. Twenty-eight days post<br />
infection, pigs were euthanised, blood sampled <strong>and</strong> a lung lesion score was given.<br />
Lung samples were processed for histopathology, immun<strong>of</strong>luorescence testing for M.<br />
hyopneumoniae <strong>and</strong> isolation <strong>of</strong> M. hyopneumoniae. RAPD analysis was performed<br />
on all M. hyopneumoniae isolates.<br />
Significant differences between isolates were found for the RDS, lung lesion<br />
score, histopathology, immun<strong>of</strong>luorescence <strong>and</strong> serology. Based on the results <strong>of</strong> the<br />
different parameters, isolates were divided into 3 “virulence” groups: low, moderately<br />
<strong>and</strong> highly virulent isolates. Typically, a 5000 bp RAPD fragment was associated with<br />
the highly <strong>and</strong> moderately virulent isolates whereas it was absent in low virulent<br />
isolates. It was concluded that high variation in virulence exists between M.<br />
hyopneumoniae isolates isolated from different swine herds. Further studies are<br />
required to determine whether the 5000 bp fragment obtained in the RAPD analysis<br />
can be used as a virulence marker.<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
INTRODUCTION<br />
Enzootic pneumonia in pigs, with M. hyopneumoniae as primary agent, is a<br />
chronic respiratory disease that causes major economic losses to the pig industry<br />
worldwide. The role <strong>of</strong> management <strong>and</strong> housing conditions in the development <strong>of</strong><br />
enzootic pneumonia has been shown to be very important (Maes et al., 1999; Maes et<br />
al., 2000). Additional factors, different from housing <strong>and</strong> management conditions may<br />
also determine the infection pattern <strong>of</strong> M. hyopneumoniae <strong>and</strong> the clinical course <strong>of</strong><br />
the infection (Vicca et al., 2002) since good conditions do not automatically result in<br />
a restricted course <strong>of</strong> enzootic pneumonia <strong>and</strong> poor conditions do not always result in<br />
severe disease.<br />
Several authors have demonstrated antigenic or genetic heterogeneity between<br />
M. hyopneumoniae isolates. Ro <strong>and</strong> Ross (1983) were able to demonstrate some<br />
diversity in antigenic structure; a different number <strong>of</strong> passages could cause a different<br />
pathogenicity in pigs (Zielinski <strong>and</strong> Ross, 1990). Frey et al. (1992) <strong>and</strong> Artiushin <strong>and</strong><br />
Minion (1996) demonstrated genetic heterogeneity by means <strong>of</strong> restriction enzyme<br />
digestion <strong>and</strong> field inversion gel electrophoresis <strong>and</strong> RAPD, respectively. However, it<br />
is not known whether genetic heterogeneity correlates with differences in virulence.<br />
The aim <strong>of</strong> this study was to assess the virulence <strong>of</strong> M. hyopneumoniae<br />
isolates obtained from different herds (Vicca et al., 2002), using a well-st<strong>and</strong>ardised<br />
experimental infection model in conventional M. hyopneumoniae- <strong>and</strong> PRRSV-free<br />
pigs. RAPD analysis was used to determine the extent <strong>of</strong> genetic variability in the M.<br />
hyopneumoniae field isolates.<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
MATERIALS AND METHODS<br />
M. hyopneumoniae isolates<br />
Six M. hyopneumoniae isolates obtained from the lungs <strong>of</strong> pigs from different<br />
Belgian farrow-to-finish herds were compared. Isolates 1, 2 <strong>and</strong> 3 were obtained from<br />
the lungs <strong>of</strong> pigs produced in herds that experienced a clinical course <strong>of</strong> enzootic<br />
pneumonia (chronic coughing in nursery <strong>and</strong> grow-finishing pigs, reduced production<br />
parameters in grow-finishing pigs). Isolates 4, 5 <strong>and</strong> 6 were obtained from herds with<br />
a subclinical course <strong>of</strong> enzootic pneumonia (no coughing or only some mild coughing<br />
in the last weeks <strong>of</strong> the grow-finishing period <strong>and</strong> the production parameters in the<br />
grow-finishing pigs reached target levels). All isolates originated from a different<br />
herd. Strain MP143 (kindly provided by Pr<strong>of</strong>. Dr. N. Friis, SVS, Denmark) was<br />
isolated in Denmark from the lungs <strong>of</strong> a pig with mycoplasmal disease.<br />
Experimental design <strong>and</strong> animals<br />
Ninety, 3-week-old, cross-bred (Seghers hybrid, Belgium), castrated male pigs<br />
were obtained from a commercial herd that had no serological evidence <strong>of</strong> exposure<br />
to M. hyopneumoniae or porcine reproductive <strong>and</strong> respiratory syndrome virus<br />
(PRRSV). The source herd was a high health breeding herd in which repeated<br />
serological monitoring <strong>of</strong> sows <strong>and</strong> pigs <strong>of</strong> different age categories has been<br />
performed during the last 5 years. To detect antibodies against M. hyopneumoniae, the<br />
DAKO ® Mh ELISA (DAKO, Glostrup, Denmark) (Feld et al. 1992) was used. During<br />
at least five years, there was no clinical evidence <strong>of</strong> M. hyopneumoniae <strong>and</strong> no lung<br />
lesions typical for M. hyopneumoniae in slaughter pigs.<br />
The pigs were weaned at 21 days <strong>of</strong> age <strong>and</strong> moved to the animal facilities at<br />
the Faculty <strong>of</strong> Veterinary Medicine, Ghent University, Belgium. They were fed a<br />
commercial feed not containing antibiotics. Animals were weighed at 4 weeks <strong>of</strong> age,<br />
ranked according to weight <strong>and</strong> stratified into 3 groups. Within each weight group,<br />
pigs were r<strong>and</strong>omly assigned to one <strong>of</strong> eight treatment groups. Each group <strong>of</strong> 10<br />
piglets was housed in rooms equipped with absolute filters (HEPA U15) to avoid<br />
spread <strong>of</strong> M. hyopneumoniae between groups.<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Immediately after weighing <strong>and</strong> allocation to the treatment groups, all pigs<br />
were anaesthetized with 0.22 ml/kg <strong>of</strong> a mixture <strong>of</strong> Xylm ® 2% (Intervet) <strong>and</strong> Zoletil<br />
100 ® (Virbac), ear tagged <strong>and</strong> inoculated intratracheally with 7 x 10 7 CCU <strong>of</strong> a M.<br />
hyopneumoniae isolate in 7 ml inoculum (groups inoculated with isolates 1-6 <strong>and</strong><br />
isolate MP143) or with 7 ml sterile culture medium (negative control group). The<br />
group inoculated with strain MP143 served as positive control group. Because <strong>of</strong> the<br />
limited number <strong>of</strong> animal rooms equipped with absolute filters, the experiment was<br />
performed during 2 successive periods. A negative control group was used during<br />
both periods, resulting in a total <strong>of</strong> 20 negative control animals. The two negative<br />
control groups were considered as one group in the statistical analyses. The<br />
inoculation day was designated as day 0 post infection (0 DPI).<br />
The study was conducted after approval by the Ethical Committee for<br />
animal experiments <strong>of</strong> the Faculty <strong>of</strong> Veterinary Medicine, Ghent University.<br />
Clinical <strong>and</strong> performance parameters<br />
For 28 days following challenge, pigs were observed daily for 25 minutes, to<br />
evaluate body condition, appetite, presence <strong>of</strong> dyspnea <strong>and</strong> tachypnea <strong>and</strong> behavioral<br />
changes. In addition, a clinical respiratory disease score (RDS) was assessed daily<br />
from 0 to 28 days post infection (DPI) (Halbur et al., 1996). RDS scores could range<br />
from 0 to 6: 0 (no coughing), 1 (mild coughing after an encouraged move), 2 (mild<br />
coughing while leaving the pigs undisturbed), 3 (moderate coughing after encouraged<br />
move), 4 (moderate coughing while leaving the pigs undisturbed), 5 (severe coughing<br />
after encouraged move), 6 (severe coughing while leaving the pigs undisturbed). The<br />
number <strong>of</strong> coughing days was assessed as the total <strong>of</strong> days on which at least one pig<br />
was coughing. Rectal temperatures were measured on a daily basis from 0-10 DPI <strong>and</strong><br />
every other day from 12-28 DPI.<br />
To calculate average daily weight gain (ADG), pigs were weighed every<br />
week, the first weighing took place at 0 DPI, the last weighing at 28 DPI (5 times in<br />
total). The amount <strong>of</strong> feed consumed during the trial (0-28 DPI) was measured to<br />
calculate the feed conversion ratio (FCR).<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Necropsy, lung lesions, immun<strong>of</strong>luorescence <strong>and</strong> bacteriological examinations<br />
All pigs were euthanised at 28 DPI by deep anesthesia with 0.3 ml/kg <strong>of</strong> a<br />
mixture <strong>of</strong> Xylm ® 2% (Intervet) <strong>and</strong> Zoletil 100 ® (Virbac) followed by<br />
exsanguination. The lungs were removed <strong>and</strong> the macroscopic pneumonic lesions<br />
were quantified using a lung lesion score diagram (Hannan et al., 1982). Total lung<br />
scores could vary between score 0 (no lesions) <strong>and</strong> a theoretical maximum <strong>of</strong> 35. A<br />
digital picture was taken from every lung for future reference.<br />
Lung tissue samples from every lobe were collected from the edge <strong>of</strong> a lung<br />
lesion, if present, <strong>and</strong> processed in a semi-quantitative immun<strong>of</strong>luorescence assay<br />
(score 0-3) to detect the presence <strong>of</strong> M. hyopneumoniae (Kobisch et al., 1978); 0: (no<br />
immun<strong>of</strong>luorescence), 1, 2 <strong>and</strong> 3 (limited, moderate <strong>and</strong> intense<br />
immun<strong>of</strong>luorescence). One score was given per lung lobe.<br />
Tissue samples from every lobe were also taken for histopathology. The tissue<br />
was fixed in 10% neutral buffered formalin <strong>and</strong> routinely processed <strong>and</strong> embedded in<br />
paraffin. The percentage <strong>of</strong> lung area occupied by air (percentage air) was examined<br />
using an automatic image analysis system (Optimas ® 6.5, Media Cybernetics, Silver<br />
Spring, USA), measuring the percentage <strong>of</strong> area occupied by air in the microscopic<br />
field (multiplication 400X). This percentage is inversely proportionate to the number<br />
<strong>of</strong> infiltrated cells, the amount <strong>of</strong> atelectasis, intrabronchiolar-intrabronchial exudate,<br />
intra-alveolar exudate <strong>and</strong>/or edema <strong>and</strong> proliferation <strong>of</strong> type II cells. An average<br />
percentage per inoculation group was calculated. This average was based on 700<br />
measurements: 10 microscopic fields per lung lobe, 7 lung lobes per lung <strong>and</strong> 10 pigs<br />
per inoculation group. The measurements were expressed as a percentage that could<br />
range from 0% through 100%.<br />
Using light microscopy, the severity <strong>of</strong> peribronchiolar <strong>and</strong> perivascular<br />
lymphohistiocytic infiltration <strong>and</strong> nodule formation consistent with M.<br />
hyopneumoniae induced pneumonia lesions were scored. The following classes were<br />
used: 0 (no cellular infiltrates), 1 (limited cellular infiltrates (macrophages <strong>and</strong><br />
lymphocytes) around bronchioles, with airways <strong>and</strong> alveolar spaces free <strong>of</strong> cellular<br />
exudates), 2 (light to moderate infiltrates with mild diffuse cellular exudates into<br />
airways), 3, 4 <strong>and</strong> 5 (mild, moderate <strong>and</strong> severe lesions characteristic <strong>of</strong> bronchointerstitial<br />
pneumonia, centered around bronchioles but extending to the interstitium,<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
with lymph<strong>of</strong>ollicular infiltration <strong>and</strong> mixed inflammatory cell exudates) (Morris et<br />
al., 1995). An average score per inoculation group was calculated, based on 700<br />
measurements: 10 measurements per lung lobe, 7 lung lobes per lung <strong>and</strong> 10 pigs per<br />
inoculation group.<br />
For each inoculation group, pooled samples <strong>of</strong> 20 percent (w/v) suspensions <strong>of</strong><br />
lungs were made, inoculated on Columbia agar supplemented with 5 percent sheep<br />
blood (blood agar) <strong>and</strong> incubated at 37°C for 48 hours to check the presence <strong>of</strong><br />
secondary bacteriological organisms in the lungs. The plates were cross-streaked with<br />
Staphylococcus aureus for support <strong>of</strong> Actinobacillus pleuropneumoniae growth.<br />
M. hyopneumoniae isolation<br />
For each inoculation group, pooled samples <strong>of</strong> 20 percent (w/v) suspensions <strong>of</strong><br />
lungs in phosphate buffered saline solution were used for isolation <strong>of</strong> M.<br />
hyopneumoniae. Isolation <strong>and</strong> cultivation <strong>of</strong> M. hyopneumoniae was optimised using<br />
earlier reports (Friis 1971a,b; 1975; 1979). Briefly, lung suspensions were inoculated<br />
into selective Friis medium. A series <strong>of</strong> subcultures in broth media were performed,<br />
followed by growing M. hyopneumoniae colonies on solid media. The mycoplasma<br />
species (M. hyopneumoniae, M. hyorhinis or M. flocculare) growing in broth media or<br />
on solid media were identified using a species specific PCR (Mattsson et al., 1995;<br />
Caron et al., 2000). For identifying M. flocculare species following primers were used<br />
(Stakenborg et al., 2005):<br />
MYCO REV<br />
AGAGGCATGATGATTTGACGTC<br />
M FLOC 741FOR TTAGGTAGGGAATGATCTAATC<br />
Single M. hyopneumoniae colonies were picked up from plate <strong>and</strong> transferred<br />
to broth medium.<br />
Serology for M. hyopneumoniae<br />
A blood sample <strong>of</strong> every pig was taken at 0 <strong>and</strong> 28 DPI to detect antibodies<br />
against M. hyopneumoniae, using the DAKO ® Mh ELISA (DAKO, Glostrup,<br />
Denmark) (Feld et al., 1992). Sera with optical density (OD)-values
2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
OD-values were considered doubtful <strong>and</strong> classified as negative in the statistical<br />
analysis.<br />
R<strong>and</strong>omly amplified polymorphic DNA (RAPD)<br />
RAPD analysis was performed on all M. hyopneumoniae isolates. The<br />
protocol used was mainly based on the article <strong>of</strong> Artiushin <strong>and</strong> Minion (1996).<br />
Briefly, 45 amplification cycles (1 min. at 95°C; 1 min. at 36°C; <strong>and</strong> 2 min. at 72°C)<br />
were run on a GeneAmp 2400 Thermal Cycler (Perkin Elmer, USA). The DNA <strong>of</strong> the<br />
M. hyopneumoniae isolates used was firstly purified using a DNA easy Kit<br />
(Westburg, The Netherl<strong>and</strong>s) to increase reproducibility. The 30 µl-reaction mixture<br />
was optimised containing 30 ng <strong>of</strong> purified genomic DNA, 2 mM MgCl 2 , 20 pmol <strong>of</strong><br />
OPA-3 primer, 120 uM dNTPs, 1x Amplitaq Buffer, 1x St<strong>of</strong>fel Buffer, 1.5 U<br />
AmpliTaq DNA polymerase <strong>and</strong> 0.75 U Amplitaq DNA polymerase St<strong>of</strong>fel Fragment<br />
(Perkin Elmer). The amplified fragments were seperated using st<strong>and</strong>ard gelelectrophoresis<br />
technique (90V, for 2 hours in TBE buffer). Smartladder (Eurogentec,<br />
Belgium) was used as size-marker. The b<strong>and</strong>s were visualized <strong>and</strong> analysed with<br />
Bionumerics s<strong>of</strong>tware (Applied Maths, Belgium).<br />
Statistical analyses<br />
Analysis <strong>of</strong> variance (ANOVA) was used to compare the results <strong>of</strong> the<br />
different inoculation groups <strong>and</strong> to link the presence or absence <strong>of</strong> a 5000bp fragment<br />
determined in RAPD to the results <strong>of</strong> the different M. hyopneumoniae inoculation<br />
groups. A Bonferroni correction was used to adjust the observed significance level for<br />
the fact that multiple comparisons were made. To estimate correlations between<br />
parameters, a bivariate analysis with Spearman’s correlation coefficient was used.<br />
The prevalence <strong>of</strong> pigs positive for the different parameters was compared<br />
between inoculation groups by logistic regression. Statistical results were considered<br />
to be significant when P-values were lower than 0.05 (two-tailed). Data were analyzed<br />
using the statistical package SPSS 10.0 for Windows (SPSS 10, SPSS Inc. Illinois<br />
60611 USA 1999).<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
RESULTS<br />
Results are summarized in Table 1<br />
Clinical evaluation <strong>and</strong> performance parameters<br />
One <strong>of</strong> the pigs inoculated with isolate 6 died at 17 DPI because <strong>of</strong> a rupture<br />
<strong>of</strong> the urinary bladder. The average rectal temperatures were not significantly<br />
different between inoculation groups (individual temperatures ranged from 38.2-41.2<br />
°C). Rectal temperatures slowly decreased towards the end <strong>of</strong> the study in all groups.<br />
Coughing was present in all groups inoculated with M. hyopneumoniae but was rarely<br />
observed in the negative control group (Figure 1). Coughing started at approximately<br />
7 DPI <strong>and</strong> increased steadily towards the end <strong>of</strong> the study. The incubation period<br />
varied according to the isolate used. The average RDS was significantly higher<br />
(P
2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
3<br />
2.5<br />
2<br />
Average RDS<br />
1.5<br />
1<br />
0.5<br />
0<br />
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28<br />
DPI<br />
Negative Control Positive Control 1<br />
2 3 4<br />
5 6<br />
Figure 1: Average respiratory disease Score (RDS) from -2 DPI until 28 days post<br />
inoculation (DPI) for pigs inoculated with sterile culture medium (negative control),<br />
strain MP143 (positive control) or Belgian M. hyopneumoniae field isolates (1-6).<br />
Macroscopic lung lesions <strong>and</strong> bacteriological examinations<br />
The lung lesion scores <strong>of</strong> pigs inoculated with strain MP143 or isolate 1, were<br />
significantly higher compared to the negative control group (P
2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Potential secondary pathogens as Pasteurella multocida, Bordetella<br />
bronchiseptica <strong>and</strong> Streptococcus suis were found in lungs <strong>of</strong> pigs in all inoculation<br />
groups, including the negative control group. The prevalences <strong>of</strong> pig lungs infected<br />
with potential secondary pathogens were 30%, 10%, 30%, 20%, 50%, 50%, 50% <strong>and</strong><br />
20% for the negative control group, positive control group <strong>and</strong> groups 1 to 6,<br />
respectively. Actinobacillus pleuropneumoniae was not isolated from any <strong>of</strong> the pig<br />
lungs. The proportions <strong>of</strong> pigs infected with secondary pathogens were not<br />
significantly different between the inoculation groups.<br />
Microscopic lung lesions<br />
Compared to the negative control group <strong>and</strong> groups inoculated with isolates 2,<br />
4 <strong>and</strong> 6, a significantly (P
2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Immun<strong>of</strong>luorescence (IF) <strong>and</strong> isolation<br />
M. hyopneumoniae organisms were not detected in the negative control group.<br />
The positive control group <strong>and</strong> the group inoculated with isolate 1 showed a<br />
significantly (P
2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
the group inoculated with isolate 2 (P
2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Correlation <strong>of</strong> parameters<br />
The parameters used to compare the virulence <strong>of</strong> the different M.<br />
hyopneumoniae isolates correlated well (r > 0.56) (Table 2). Percentage air <strong>and</strong><br />
serology (OD-values) were negatively correlated with the other parameters (RDS,<br />
number <strong>of</strong> coughing days, lung lesions, lymphohistiocyte infiltration <strong>and</strong><br />
immun<strong>of</strong>luorescence).<br />
Table 2: Spearman’s correlation coefficients <strong>of</strong> parameters used to evaluate virulence<br />
<strong>of</strong> the M. hyopneumoniae field isolates<br />
Respiratory<br />
Disease<br />
Score<br />
Number<br />
<strong>of</strong><br />
coughing<br />
days<br />
Lymphohistiocytic<br />
infiltration<br />
Percentage<br />
air<br />
Immun<strong>of</strong>luorescence<br />
Lung<br />
lesions<br />
Respiratory<br />
1.000 0.991 0.662 - 0.643 0.755 0.804 - 0.736<br />
Disease Score<br />
Number <strong>of</strong><br />
1.000 0.644 - 0.636 0.769 0.811 - 0.735<br />
coughing days<br />
Lymphohistiocytic<br />
1.000 - 0.604 0.705 0.670 - 0.704<br />
infiltration<br />
Percentage air 1.000 - 0.604 - 0.604 0.564<br />
Serology<br />
(ODvalues)<br />
Immun<strong>of</strong>luorescence<br />
1.000 0.893 - 0.780<br />
Lung lesions 1.000 - 0.815<br />
Serology<br />
(OD-values)<br />
1.000<br />
All correlations were significant at the 0.01 level<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
DISCUSSION<br />
This study proved the hypothesis that differences in virulence between<br />
circulating M. hyopneumoniae field isolates exist. This hypothesis was based on<br />
observed different clinical courses <strong>of</strong> the disease in the herds selected for isolation <strong>of</strong><br />
the isolates. Although the importance <strong>of</strong> management <strong>and</strong> housing conditions has<br />
been shown several times (Clark et al., 1991; Ross, 1999) <strong>and</strong> should never be<br />
underestimated, earlier field experiments performed at our department (Vicca et al.,<br />
2002) showed that additional factors different from housing <strong>and</strong> management<br />
conditions may determine the infection pattern <strong>of</strong> M. hyopneumoniae <strong>and</strong> the clinical<br />
course <strong>of</strong> the infection.<br />
It has been shown that a different number <strong>of</strong> in vitro passages can result in a<br />
different pathogenicity <strong>of</strong> M. hyopneumoniae isolates in pigs (Whittlestone, 1979;<br />
Zielinsky <strong>and</strong> Ross, 1990). In the present studies, the number <strong>of</strong> passages used to<br />
obtain pure cultures <strong>of</strong> isolates 1, 3, 4, 5 <strong>and</strong> 6 varied between 8 <strong>and</strong> 10,<br />
demonstrating that differences in virulence between these isolates were not due to<br />
different numbers <strong>of</strong> in vitro passages. Isolate 2 was passed 20 times in vitro. It<br />
cannot be excluded that the higher number <strong>of</strong> passages played a role in decreased<br />
virulence <strong>of</strong> this isolate.<br />
Performance parameters such as ADG <strong>and</strong> FCR were <strong>of</strong> little use in this study<br />
to evaluate differences in virulence. Although these parameters are very important to<br />
evaluate the economical results <strong>of</strong> a swine herd, the number <strong>of</strong> animals used <strong>and</strong> the<br />
duration <strong>of</strong> the study period were probably too limited to observe an effect <strong>of</strong> the M.<br />
hyopneumoniae inoculation on these performance parameters.<br />
The parameters RDS, lung lesion score, infiltration <strong>of</strong> lymphohistiocytes,<br />
percentage <strong>of</strong> air left in the lung, immun<strong>of</strong>luorescence <strong>and</strong> serology appeared to be<br />
important to evaluate the virulence <strong>of</strong> isolates during this study. A good correlation<br />
between the above-mentioned parameters was demonstrated (Table 2). Kobisch et al.<br />
(1993) <strong>and</strong> Morris et al. (1995) already found a positive correlation between coughing<br />
<strong>and</strong> lung lesion scores. In an earlier study from Zielinsky <strong>and</strong> Ross (1990), no<br />
consistent relation between microscopic lesions <strong>and</strong> the extent <strong>of</strong> macroscopic lesions<br />
was found; this is in contradiction with our results. Zielinsky <strong>and</strong> Ross (1990)<br />
suggested that the extent <strong>of</strong> macroscopic lesions was rather influenced by natural<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
susceptibility (attributable to genetic components) <strong>of</strong> the pigs than by the action <strong>of</strong> the<br />
organism itself. All pigs used in our study were born in the same herd, a breeding herd<br />
that strives for genetic homogeneity. The genetic variability between pigs could not<br />
have been sufficiently large to be the only explanation for the observed variability in<br />
extent <strong>of</strong> macroscopic lesions between inoculation groups.<br />
Based on the outcomes <strong>of</strong> the clinical <strong>and</strong> pathological parameters, the isolates<br />
could roughly be divided into 3 groups: highly virulent isolates (strain MP143 <strong>and</strong><br />
isolate 1), moderately virulent isolates (3 <strong>and</strong> 5) <strong>and</strong> low virulent isolates (2, 4 <strong>and</strong> 6).<br />
For all parameters, highly virulent isolates differed significantly from the negative<br />
control group. Low virulent isolates did not show significant differences from the<br />
negative control group, but did show significant differences from the highly virulent<br />
isolates for the parameters: RDS, lung lesion score, percentage air, lymphohistiocytic<br />
infiltration (except isolate 2) <strong>and</strong> IF (except isolate 4). The isolates 3 <strong>and</strong> 5 could<br />
statistically not be distinguished clearly from the other groups: the results <strong>of</strong> RDS <strong>and</strong><br />
macroscopic lung lesions were more in accordance with the results <strong>of</strong> the low virulent<br />
isolates, the results from the microscopic lesions, IF <strong>and</strong> serology were more in<br />
accordance with the results <strong>of</strong> the highly virulent isolates. Therefore, these isolates<br />
were considered as moderately virulent. Since microscopic lesions due to M.<br />
hyopneumoniae infection are evident at an earlier time than macroscopic lesions, a<br />
longer study period (> 28 days) could have resulted in a more severe RDS <strong>and</strong> lung<br />
lesion score.<br />
The intensity <strong>of</strong> immun<strong>of</strong>luorescence was clearly higher in the lungs <strong>of</strong> pigs<br />
inoculated with the highly virulent isolates. This might indicate that the highly<br />
virulent isolates had a higher capacity to colonize or attach to the ciliated epithelium<br />
or were able to multiply faster resulting in more organisms causing an earlier <strong>and</strong><br />
more severe coughing, more severe macroscopic <strong>and</strong> microscopic lesions <strong>and</strong> more<br />
intense immun<strong>of</strong>luorescence. A difference in capability to attach to cilia between M.<br />
hyopneumoniae isolates has been demonstrated by Minion et al. (2000) <strong>and</strong> was<br />
correlated with the number <strong>of</strong> repeat regions in RR1 <strong>of</strong> the P97 protein <strong>of</strong> M.<br />
hyopneumoniae. A minimum <strong>of</strong> eight repeats was necessary to make the M.<br />
hyopneumoniae organisms able to attach to the cilia. Research on the P97 protein <strong>of</strong><br />
the different M. hyopneumoniae isolates used in this study could provide important<br />
information about the attachment capacities <strong>of</strong> the isolates <strong>and</strong> could partly explain<br />
the differences in virulence between isolates observed in this study.<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Piglets inoculated with a highly virulent isolate had a higher number <strong>of</strong> piglets<br />
seroconverted at 28 DPI compared to piglets inoculated with a low virulent isolate.<br />
An earlier seroconversion <strong>of</strong> 3 weeks was also seen in herds experiencing a clinical<br />
course <strong>of</strong> enzootic pneumonia compared to herds subclinically infected with M.<br />
hyopneumoniae (Vicca et al., 2002). The reason for this finding is not clear but it<br />
might be because <strong>of</strong> a higher immunogenicity or a faster multiplication <strong>of</strong> the highly<br />
virulent isolates. A difference in glycosylation <strong>of</strong> immunogenic proteins between M.<br />
hyopneumoniae isolates was described by Lin (2001), but no relation to virulence <strong>of</strong><br />
the isolates was made. Differences in immunogeneity between different M.<br />
hyopneumoniae isolates could be <strong>of</strong> special interest for future vaccine development.<br />
A 5000 bp RAPD b<strong>and</strong> was only present in highly <strong>and</strong> moderately virulent<br />
isolates <strong>and</strong> not in low virulent isolates. Further research is required to determine<br />
whether this b<strong>and</strong> can be used as a virulence marker for M. hyopneumoniae.<br />
CONCLUSION<br />
In the present study, variation in virulence between different field isolates <strong>of</strong><br />
M. hyopneumoniae was demonstrated in an experimental infection model. Further<br />
studies are required to determine the usefulness <strong>of</strong> the 5000 bp RAPD b<strong>and</strong> to predict<br />
virulence <strong>of</strong> M. hyopneumoniae isolates.<br />
ACKNOWLEDGEMENTS<br />
This research was financially supported by grant S-5940 (section II) <strong>of</strong> the<br />
Belgian Ministry <strong>of</strong> Small Enterprises, Trader <strong>and</strong> Agriculture, Research Foundation<br />
(Brussels). I am grateful to all the people who assisted me with the practical aspects <strong>of</strong><br />
this study.<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
REFERENCES<br />
1. Artiushin, S. & Minion, F.C. (1996). Arbitrarily primed PCR analysis <strong>of</strong> Mycoplasma<br />
hyopneumoniae field isolates demonstrates genetic heterogeneity. International Journal <strong>of</strong><br />
Systemic Bacteriology 46, 324-328.<br />
2. Caron, J., Ouardani, M. & Dea, S. (2000). Diagnosis <strong>and</strong> differentiation <strong>of</strong> Mycoplasma<br />
hyopneumoniae <strong>and</strong> Mycoplasma hyorhinis infections in pigs by PCR amplification <strong>of</strong> the p36<br />
<strong>and</strong> p46 genes. Journal <strong>of</strong> Clinical Microbiology 38, 1390-1396.<br />
3. Clark, L., Armstrong, C., Knox, K. & Mayrose, V. (1991). The effect <strong>of</strong> all-in/all-out<br />
management on pigs from a herd with enzootic pneumonia. Veterinary Medicine 86, 946-951.<br />
4. Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F. & Meyling, A. (1992). A monoclonal blocking<br />
ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. Veterinary Microbiology 30,<br />
35-46.<br />
5. Frey, J., Haldimann, A. & Nicolet, J. (1992). Chromosomal heterogeneity <strong>of</strong> various<br />
Mycoplasma hyopneumoniae field strains. International Journal <strong>of</strong> Systemic Bacteriology 42,<br />
275-280.<br />
6. Friis, N.F. (1971ª). A selective medium for Mycoplasma suipneumoniae. Acta Veterinaria<br />
Sc<strong>and</strong>inavia 12, 454-456.<br />
7. Friis, N.F. (1971b). Mycoplasmas cultivated from the respiratory tract <strong>of</strong> Danish pigs. Acta<br />
Veterinaria Sc<strong>and</strong>inavia 12, 69-79.<br />
8. Friis, N.F. (1975). Some recommendations concerning primary isolation <strong>of</strong> Mycoplasma<br />
suipneumoniae <strong>and</strong> Mycoplasma flocculare. Nordisk Veterinaer Medicin 27, 337-339.<br />
9. Friis, N.F. (1979). Selective isolation <strong>of</strong> slowly growing acidifying mycoplasmas from swine<br />
<strong>and</strong> cattle. Acta Veterinaria Sc<strong>and</strong>inavia 20, 607-609.<br />
10. Halbur, P.G., Paul, P.S., Meng, X.J., Lum, M.A., Andrews, J.J. & Rathje, J.A. (1996).<br />
Comparative pathogenicity <strong>of</strong> nine US Porcine Reproductive <strong>and</strong> Respiratory Syndrome Virus<br />
(PRRSV) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model. Journal <strong>of</strong><br />
Veterinary Diagnostic Investigation 8, 11-20.<br />
11. Hannan, P.C., Bhogal, B.S. & Fish, J.P. (1982). Tylosin tartrate <strong>and</strong> tiamutilin effects on<br />
experimental piglet pneumonia induced with pneumonic pig lung homogenate containing<br />
mycoplasmas, bacteria <strong>and</strong> viruses. Research in Veterinary Science 33, 76-88.<br />
12. Kobisch, M., Tillon, J.P., Vannier, Ph., Magueur, S. & Morvan, P. (1978). Pneumonie<br />
enzootique à Mycoplasma suipneumoniae chez le porc: diagnostic rapide et recherche<br />
d’anticorps. Recueil de Medecine Veterinaire 154, 847-852.<br />
13. Kobisch, M., Blanchard, B. & Le Potier, M.F. (1993). Mycoplasma hyopneumoniae infection<br />
in pigs: duration <strong>of</strong> the disease <strong>and</strong> resistance to reinfection. Veterinary Research 24, 67-77.<br />
14. Lin, B. (2001). Intraspecies differentiation <strong>of</strong> Mycoplasma hyopneumoniae field strains isolated<br />
in the United States. American Association <strong>of</strong> Swine Veterinarians, 225-235.<br />
15. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A.<br />
(1999). Risk indicators for the seroprevalence <strong>of</strong> Mycoplasma hyopneumoniae, porcine<br />
Influenza viruses <strong>and</strong> Aujeszky’s disease virus in slaughter pigs from fattening pig herds.<br />
Journal <strong>of</strong> Veterinary Medicine, Series B 46, 341-352.<br />
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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
16. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A.<br />
(2000). <strong>Herd</strong> factors associated with the seroprevalences <strong>of</strong> four major respiratory pathogens in<br />
slaughter pigs from farrow-to-finish pig herds. Veterinary Research 31, 313-327.<br />
17. Mattsson, J.G., Bergström, K., Wallgren, P. & Johansson, K.E. (1995). Detection <strong>of</strong><br />
Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification <strong>of</strong> the 16s RNA<br />
gene. Journal <strong>of</strong> Clinical Microbiology 33, 893-897.<br />
18. Minion, F.C., Adams, C. & Hsu, T. (2000). R1 region <strong>of</strong> P97 mediates adherence <strong>of</strong><br />
Mycoplasma hyopneumoniae to swine cilia. Infection <strong>and</strong> Immunity 68, 3056-3060.<br />
19. Morris, C.R., Gardner, I.A., Hietala, S.K. & Carpenter, T.E. (1995). Enzootic pneumonia:<br />
comparison <strong>of</strong> cough <strong>and</strong> lung lesions as predictors <strong>of</strong> weight gain in swine. Canadian Journal <strong>of</strong><br />
Veterinary Research 59, 197-204.<br />
20. Ro, L.H. & Ross, R.F. (1983). Comparison <strong>of</strong> Mycoplasma hyopneumoniae strains by serologic<br />
methods. American Journal <strong>of</strong> Veterinary Research 11, 2087-2094.<br />
21. Ross, R.F. (1999). Mycoplasmal Diseases. In: Leman A.D., Straw B.E., Mengeling W.L.,<br />
Allaire S.D., Taylor D.J (Eds.), Diseases <strong>of</strong> Swine, 8th Edition, Iowa State University Press,<br />
Ames, pp. 537-551.<br />
22. Stakenborg, T., Vicca, J., Butaye, P., Imberechts, H., Peeters, J., de Kruif, A.,<br />
Haesebrouck, F., Maes, D. (2005). A multiplex PCR to identify mycoplasmas present in broth<br />
cultures. Veterinary Research Communications, In press.<br />
23. Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F. & de Kruif, A. (2002).<br />
Patterns <strong>of</strong> Mycoplasma hyopneumoniae infections in Belgian farrow-to-finish pig herds with<br />
diverging disease-course. Journal <strong>of</strong> Veterinary Medicine, Series B 49, 349-353.<br />
24. Whittlestone, P. (1979). Porcine mycoplasmas. In: Tully, J.G. <strong>and</strong> Whitcomb, R.F. (Ed.), The<br />
Mycoplasmas II. Human <strong>and</strong> Animal Mycoplasmas, Academic press, Inc., New York, pp. 133-<br />
176.<br />
25. Zielinsky, G. & Ross, R. (1990). Effect <strong>of</strong> growth in cell cultures <strong>and</strong> strain on virulence <strong>of</strong><br />
Mycoplasma hyopneumoniae for swine. American Journal <strong>of</strong> Veterinary Research 51, 344-348.<br />
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2.2 SUSCEPTIBILITY OF M. HYOPNEUMONIAE TO<br />
ANTIMICROBIAL AGENTS<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD<br />
ISOLATES<br />
Modified from:<br />
IN VITRO SUSCEPTIBILITIES OF MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES<br />
J. VICCA, T. STAKENBORG, D. MAES, P. BUTAYE, J. PEETERS, A. DE KRUIF & F. HAESEBROUCK<br />
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 2004, 48, 4470-4472<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
SUMMARY<br />
Mycoplasma hyopneumoniae causes enzootic pneumonia, a chronic<br />
respiratory disease in pigs. To control the infection, preventive <strong>and</strong> curative use <strong>of</strong><br />
antibiotics in feed or water is a common practice. Unfortunately, susceptibility <strong>of</strong> M.<br />
hyopneumoniae to different antimicrobials has rarely been determined. In this study, a<br />
broth microdilution technique was used to determine the in vitro susceptibilities <strong>of</strong> 21<br />
M. hyopneumoniae field isolates. Acquired resistance to spectinomycin,<br />
oxytetracycline, doxycycline, gentamicin, florfenicol <strong>and</strong> tiamulin was not observed.<br />
One isolate showed acquired resistance to lincomycin, tilmicosin <strong>and</strong> tylosin. This<br />
isolate was susceptible to all other antibiotics tested. The MIC-values <strong>of</strong> flumequine<br />
were > 16 µg/ml for 5 isolates, while the MIC 50 -value was 2 µg/ml. For these 5<br />
isolates, the MIC-values for enr<strong>of</strong>loxacin were ≥ 0.5 µg/ml, the MIC 50 being 0.06<br />
µg/ml. As far as we know, this is the first report <strong>of</strong> acquired resistance against<br />
macrolides, lincosamides <strong>and</strong> fluoroquinolones in M. hyopneumoniae field isolates.<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
INTRODUCTION<br />
Mycoplasma hyopneumoniae causes enzootic pneumonia, a chronic<br />
respiratory disease in pigs resulting in considerable economic losses. Although<br />
appropriate vaccines are available to reduce the consequences <strong>of</strong> infection, in feed or<br />
water medication with antimicrobials is still a common practice to treat or control the<br />
disease. The use <strong>of</strong> antimicrobials, however, results in the selection <strong>of</strong> resistant<br />
bacteria. Antimicrobial susceptibility <strong>of</strong> M. hyopneumoniae field isolates has rarely<br />
been determined <strong>and</strong> limited numbers <strong>of</strong> isolates have been considered in these<br />
studies (Hannan et al., 1989; Ter Laak et al., 1991; Tanner et al., 1993; Friis <strong>and</strong><br />
Szancer, 1994; Hannan et al., 1997a; Hannan et al., 1997b). This is mainly due to the<br />
fact that M. hyopneumoniae is a very fastidious <strong>and</strong> slowly growing micro-organism<br />
which makes it difficult to obtain large numbers <strong>of</strong> field isolates. In addition,<br />
susceptibility testing is more difficult for these organisms compared to other bacteria<br />
as there is no st<strong>and</strong>ardized procedure. Recently, guidelines <strong>and</strong> recommendations for<br />
MIC testing against veterinary mycoplasma species have been proposed on behalf <strong>of</strong><br />
the International Research Programme on Comparative Mycoplasmology (IRPCM)<br />
(Hannan, 2000).<br />
In the present study, antimicrobial susceptibility <strong>of</strong> M. hyopneumoniae<br />
isolates, recently obtained from swine in the field, was determined using these<br />
guidelines <strong>and</strong> recommendations. Also, antibiotic use on the herds where the isolates<br />
where obtained was monitored in order to try to find a possible link between antibiotic<br />
use <strong>and</strong> MIC results.<br />
MATERIALS AND METHODS<br />
M. hyopneumoniae isolates<br />
Twenty-one M. hyopneumoniae field isolates, obtained between 2000 <strong>and</strong><br />
2002 from the lungs <strong>of</strong> pigs from 21 different farrow-to-finish pig herds in Belgium,<br />
were used for MIC determination (Vicca et al., 2002; Vicca et al., 2003). All isolates<br />
were obtained from the lungs <strong>of</strong> slaughter pigs, except isolates 1, 4, 5 <strong>and</strong> 6 which<br />
were obtained from the lungs <strong>of</strong> 9-12 week old euthanized pigs which did not present<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
respiratory tract disease signs. The antibiotics used on the different herds in suckling<br />
piglets (0 - 4 weeks <strong>of</strong> age), nursery piglets (4 - 10 weeks <strong>of</strong> age) <strong>and</strong> growth -<br />
finishing pigs (10 weeks until slaughter) during the year before the M. hyopneumoniae<br />
isolates were obtained, are mentioned in Table 1.<br />
Isolation <strong>and</strong> cultivation <strong>of</strong> M. hyopneumoniae was optimized using earlier<br />
reports (Friis 1971a,b; 1975; 1979). Briefly, lung suspensions were inoculated into<br />
selective Friis medium. A series <strong>of</strong> subcultures in selective <strong>and</strong> non-selective Friis<br />
medium were carried out, followed by growing M. hyopneumoniae colonies on solid<br />
medium (agar mixed with non-selective Friis medium). M. hyopneumoniae was<br />
identified using a species specific PCR (Mattsson et al., 1995; Caron et al., 2000).<br />
Single M. hyopneumoniae colonies were picked up from plate <strong>and</strong> transferred<br />
to non-selective Friis medium. Isolates were stored at -70°C until used.<br />
The M. hyopneumoniae type strain ATCC 25634 (J-strain) was used as control strain.<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Table 1: The antibiotics used during 1 year before isolation <strong>of</strong> M. hyopneumoniae on<br />
the herd<br />
<strong>Herd</strong> Isolate Antibiotics b used in<br />
Suckling piglets Nursery piglets Grow-finishing pigs<br />
A 1 ENRO 1, a ENRO 1 OXY 1<br />
B 2 DANO 1 COL 5 none<br />
C 4* ENRO 1 COL 5 TYL 4 or PEN 1<br />
D 5 AMK 2 AMK+COL 5 OXY 3 or DOX 3<br />
E 6 AMK 2 or PEN 2 TMP+SULF 3 TYL 3 or DOX 3<br />
F 7 ENRO 1 COL 4 , APR 4 , AMK 4 , None<br />
OXY 4 , ENRO 1<br />
G 8* PEN 2 , ENRO 1 , NEO 1 LIN 1 , OXY 6 , TYL 6 LIN 1 , OXY 6 , TYL 6<br />
H 9 AMK 2 LIN 1 , OXY 6 LIN 1 , OXY 6<br />
I 10 none COL 3 none<br />
J 11 none AMK 3 none<br />
K 12 none COL 5 none<br />
L 13<br />
AMK 1 , ENRO 1 , PEN 2 AMK 1 , APR 3 , COL 3 ,<br />
DOX 4 , ENRO 1 ,<br />
CEF 1 , TYL 1<br />
TMP+SULF 3 ,<br />
M 14* ENRO1, PEN+STR 1 , AMK 5 , COL 5 , ENRO 2 , OXY 5<br />
CEF 1 , AMK 1 CEF 1 , PEN+STR 1<br />
N 15 none none TMP+SULF 6<br />
O 16 TMP+SULF 1 , AMK 1 OXY 1 OXY 1<br />
P 17* AMK 1 , ENRO 1 AMK 5 , AMK 1 DOX 5 , FFN 1<br />
Q 18 CEF 1 , ENRO 1 COL 5 , TMP+SULF 4 none<br />
R 19** none none LIN 1<br />
S 20* ENRO 1 ENRO 1 , FLUM 3 ENRO 1 , PEN 1 , OXY 1<br />
T 21 none none DOX 4<br />
U 23 COL 1 , AMK 2 COL 5 TYL+DOX 5<br />
a :<br />
1 : injectable (some pigs injected in case <strong>of</strong> disease); 2 : injectable (all pigs injected, routine ); 3 :<br />
in feed medication (routine); 4 : in feed medication (in case <strong>of</strong> disease); 5 : in water medication<br />
(routine); 6 : in water medication (in case <strong>of</strong> disease)<br />
b<br />
: AMK, amoxicillin; APR, apramycin; CEF, cefquinome; COL, colistin; DANO, dan<strong>of</strong>loxacin;<br />
DOX, doxycycline; ENRO, enr<strong>of</strong>loxacin; FFN, florfenicol; FLUM, flumequine; LIN,<br />
lincomycin; OXY, oxytetracycline; PEN, penicillin; PEN+STR, penicillin <strong>and</strong> streptomycin;<br />
TMP+SULF, trimethoprim <strong>and</strong> sulfamides; TYL, tylosin<br />
*: High MIC values for flumequine <strong>and</strong> enr<strong>of</strong>loxacin<br />
**: High MIC values for tylosin, tilmicosin <strong>and</strong> lincomycin<br />
Minimal inhibitory concentration (MIC) determination<br />
Ninety-six well, round-bottom microtitre plates (Sensititre Ltd., Imberhorne<br />
Lane, East Grinstead, Sussex, Engl<strong>and</strong>) containing stabilized freeze-dried lincomycin<br />
(0.06 - 8 µg/ml), lincomycin / spectinomycin 1:2 ratio (0.06/0.12 - 8/16 µg/ml),<br />
spectinomycin (0.12 - 16 µg/ml), oxytetracycline (0.015 - 2 µg/ml), doxycycline<br />
(0.015 - 2 µg/ml), enr<strong>of</strong>loxacin (0.008 - 1 µg/ml), flumequine (0.12 - 16 µg/ml),<br />
gentamicin (0.12 - 16 µg/ml), florfenicol (0.12 - 16 µg/ml), tiamulin (0.015 - 1<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
µg/ml), tilmicosin (0.25 - 16 µg/ml) <strong>and</strong> tylosin tartrate (0.015 - 1 µg/ml) were used.<br />
Three wells on each plate were left antimicrobial-free as a positive growth control.<br />
M. hyopneumoniae isolates were removed from cryostorage (-70°C), allowed<br />
to thaw at room temperature <strong>and</strong> incubated during 1-2 hours in non-selective Friis<br />
medium at 36 ± 1 °C. Isolates were vortexed <strong>and</strong> diluted in non-selective Friis<br />
medium until the number <strong>of</strong> organisms reached 10 4 CCU/ml. Fifty µl <strong>of</strong> the diluted<br />
culture was transferred into each well <strong>of</strong> the Sensititre® plate. The J-strain was tested<br />
3 times in order to estimate reproducibility <strong>of</strong> the procedure. After inoculation, the<br />
microtiter plates were sealed using an adhesive foil <strong>and</strong> incubated at 36 ± 1 °C for 14<br />
days. Plates were observed daily. Growth <strong>of</strong> M. hyopneumoniae organisms was<br />
observed when the colour <strong>of</strong> the medium changed from red to yellow (phenol red<br />
indicator), corresponding with a decrease in pH from 7.4 to 6.8 by glucose<br />
fermentation. The initial <strong>and</strong> final MICs were recorded. The initial MIC was defined<br />
as the lowest antibiotic concentration to show no change in colour when the colour <strong>of</strong><br />
the growth control turned yellow. The final MIC was defined as the lowest antibiotic<br />
concentration to show no change in colour at 14 days after inoculation (Tanner <strong>and</strong><br />
Wu, 1992).<br />
RESULTS<br />
In Table 2, the initial <strong>and</strong> final MIC 50 , MIC 90 <strong>and</strong> MIC-range are presented<br />
for the 21 M. hyopneumoniae field isolates. Only for oxytetracycline, doxycycline <strong>and</strong><br />
gentamicin the initial <strong>and</strong> final MIC 50 differed from each other for more than one<br />
dilution. No clear differences were detected between initial <strong>and</strong> final MIC 90 values.<br />
The MIC-values for the 3 replicates <strong>of</strong> the J-strain are also given in Table 2. It can be<br />
seen that these values were equal or differed from each other for only one doubling<br />
dilution, indicating a good reproducibility <strong>of</strong> the test. The initial MIC-values for the J-<br />
strain were in agreement with the values reported in earlier studies (Hannan et al.,<br />
1989; Ter Laak et al., 1991; Tanner et al., 1993; Hannan et al., 1997a,b).<br />
In Table 3, frequency distributions <strong>of</strong> final MIC values <strong>of</strong> the different<br />
antibiotics for the 21 field isolates are presented.<br />
The MICs <strong>of</strong> oxytetracycline, doxycycline, tiamulin, spectinomycin,<br />
gentamicin, florfenicol <strong>and</strong> the lincomycin - spectinomycin combination did not show<br />
any indication <strong>of</strong> a bimodal frequency distribution range (Butaye et al., 2003). For<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
one isolate, obtained on farm R, the MICs <strong>of</strong> tylosin, tilmicosin <strong>and</strong> lincomycin were<br />
clearly higher than for the other isolates. Isolates number 4, 8, 14, 17 <strong>and</strong> 20 had a<br />
MIC value <strong>of</strong> > 16 µg/ml for flumequine while the final MIC 50 equals only 2 µg/ml,<br />
corresponding to the median <strong>of</strong> the first distribution. For the same isolates the MIC<br />
value <strong>of</strong> enr<strong>of</strong>loxacine was ≥ 0.5 µg/ml while the MIC 50 was 0.06 µg/ml.<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Table 2: Initial <strong>and</strong> final MIC 50 , MIC 90 en MIC-range (µg/ml) <strong>of</strong> antimicrobials<br />
against Belgian M. hyopneumoniae field isolates. The MIC for the reference strain (Jstrain)<br />
was determined 3 times <strong>and</strong> the results are included is this table.<br />
Antimicrobial 1 Reading Field isolates (n=21) Reference strain:<br />
J-strain<br />
MIC 50 MIC 90 MIC-Range MIC-range<br />
LIN Initial ≤ 0.06 ≤ 0.06 ≤ 0.06 - > 8 8 0.25<br />
LIN/SPT Initial ≤ 0.06/0.12 ≤<br />
0.06/0.12<br />
≤ 0.06/0.12 -<br />
0.25/0.5<br />
Final ≤ 0.06/0.12 0.12/0.25 ≤ 0.06/0.12 -<br />
0.25/0.5<br />
2 1<br />
DOX Initial 0.12 0.5 0.03 - 1 0.06 - 0.12<br />
Final 0.5 1 0.12 - 2 0.5 - 1<br />
ENRO Initial 0.03 0.5 0.015 - > 1 0.015 - 0.03<br />
Final 0.06 0.5 0.03 - > 1 0.06<br />
FLUM Initial 1 > 16 0.25 - > 16 0.5 - 1<br />
Final 2 > 16 0.5 - > 16 2<br />
GEN Initial ≤ 0.12 0.5 ≤ 0.12 - 1 0.25 - 0.5<br />
Final 0.5 1 ≤ 0.12 - 1 0.5 - 1<br />
FFN Initial ≤ 0.12 0.25 ≤ 0.12 - 0.5 0.25<br />
Final 0.25 0.5 ≤ 0.12 - 1 1<br />
TIA Initial ≤ 0.015 0.12 ≤ 0.015 - 0.12 0.03<br />
Final 0.03 0.12 ≤ 0.015 - 0.12 0.06<br />
TIL Initial 0.25 0.5 ≤ 0.25 - > 16 0.25<br />
Final 0.5 0.5 ≤ 0.25 - > 16 0.5<br />
TYL Initial 0.03 0.06 ≤ 0.015 - > 1 1 0.12<br />
1 : LIN, lincomycin; SPT, spectinomycin; OXY, oxytetracycline; DOX, doxycycline; ENRO,<br />
enr<strong>of</strong>loxacin; FLUM, flumequine; GEN, gentamicin; FFN, florfenicol; TIA, tiamulin; TIL,<br />
tilmicosin; TYL, tylosin<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Table 3: Frequency distribution <strong>of</strong> final minimal inhibitory concentrations (MICs) <strong>of</strong><br />
12 antibiotics on 21 Belgian M. hyopneumoniae field strains.<br />
Antimicrobial 1<br />
Number <strong>of</strong> stains with MIC (µg/ml) <strong>of</strong><br />
0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16<br />
Lincomycin 16 4 3 1** (>)*<br />
Lincomycin/<br />
17 4 3<br />
Spectinomycin<br />
Spectinomycin 3 (≤ ) 2 8 11<br />
Oxytetracycline 5 2 5 5 6, 1 (>)<br />
Doxycycline 5 3 5 9 2<br />
Enr<strong>of</strong>loxacin 5 13 1 4 1 (>)<br />
Flumequine 3 3 11 2 5 (>)<br />
Gentamicin 4 (≤ ) 4 10 6<br />
Florfenicol 7 (≤ ) 8 6 3<br />
Tiamulin 4 10 7 3<br />
Tilmicosin 10 (≤ ) 13 1 (>)<br />
Tylosin 2 5 6 10 1 (>)<br />
*: > : higher than MIC indicated<br />
≤: equal or lower than MIC indicated<br />
**: isolates considered to have acquired resistance are underlined<br />
DISCUSSION<br />
In the present study a bimodal frequency distribution <strong>of</strong> MICs for the<br />
macrolides tylosin <strong>and</strong> tilmicosin as well as for the lincosamide antibiotic, lincomycin<br />
was seen. The MICs <strong>of</strong> these antibiotics were clearly higher for one isolate, indicating<br />
acquired resistance. The MIC <strong>of</strong> tylosin for this isolate was also higher than the<br />
breakpoint proposed by Hannan et al. (1997a). Macrolides <strong>and</strong> lincosamides are<br />
chemically distinct but they have a similar mode <strong>of</strong> action <strong>and</strong>, moreover, overlapping<br />
binding sides on the 23S rRNA <strong>of</strong> the 50S subunit <strong>of</strong> the bacterial ribosome. They act<br />
by blocking protein synthesis on assembled <strong>and</strong> functioning 50S ribosomal subunits<br />
(Weisblum, 1995; Butaye 2000). Acquired resistance against these antibiotics has not<br />
been described before in M. hyopneumoniae <strong>and</strong> the mechanism <strong>of</strong> resistance is<br />
unknown. In M. pneumoniae, acquired resistance against macrolides has been<br />
associated with an alteration in the drug target side by point mutations in the 23s<br />
rRNA gene (Lucier et al., 1995).<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
Acquired resistance to macrolides <strong>and</strong> lincosamides in mycoplasmas has not<br />
been reported <strong>of</strong>ten, most probably due to a limited amount <strong>of</strong> isolates tested. Only 2<br />
M. pneumoniae, 1 Ureaplasma spp. <strong>and</strong> 2 M. hominis resistant strains were isolated<br />
from humans (Bébéar <strong>and</strong> Bébéar, 2002). In animal mycoplasmas, acquired resistance<br />
to tylosin has been described for M. gallisepticum (Levisohn, 1981), M. hyosynoviae<br />
(Kobayashi et al., 1996b; Aarestrup et al. 1998), M. hyorhinis (Kobayashi et al.,<br />
1996a,b) <strong>and</strong> M. bovis (Thomas et al., 2003). No co-resistance to lincomycin was<br />
observed for the tylosin resistant M. hyosynoviae isolates. Lincomycin was not<br />
evaluated by Levisohn (1981), <strong>and</strong> although tylosin <strong>and</strong> lincomycin resistant M. bovis<br />
isolates were found by Thomas et al. (2003), the existence <strong>of</strong> co-resistance was not<br />
reported. Ter Laak et al. (1993) found M. bovis isolates that were resistant to<br />
lincomycin, but they were susceptible to tylosin. The use <strong>of</strong> lincomycin in growfinishing<br />
pigs on the herd where our macrolide-lincosamide resistant M.<br />
hyopneumoniae isolate was obtained may have contributed to selection <strong>of</strong> antibiotic<br />
resistance.<br />
The MICs <strong>of</strong> flumequine <strong>and</strong> enr<strong>of</strong>loxacin also showed an extended frequency<br />
distribution range with a tendency towards bimodality. For 5 <strong>of</strong> the 21 isolates the<br />
MIC <strong>of</strong> flumequine was >16 µg/ml, which is higher than the breakpoint <strong>of</strong> ≥ 16 µg/ml<br />
proposed by Hannan et al. (1997a). For these isolates, the MIC <strong>of</strong> enr<strong>of</strong>loxacin was ≥<br />
0.5 µg/ml while the MIC 50 was 0.06 µg/ml. This rather high frequency <strong>of</strong> acquired<br />
resistance against fluoroquinolones is unusual. Although fluoroquinolone resistant M.<br />
hominis isolates have been reported (Bébéar et al., 1999), resistance against these<br />
antibiotics has been rarely reported in human associated mycoplasmas. Hannan et al.<br />
(1997a) described acquired resistance against flumequine in avian, porcine (but not M.<br />
hyopneumoniae), bovine, ovine <strong>and</strong> caprine mycoplasmas <strong>and</strong> Thomas et al. (2003)<br />
isolated enr<strong>of</strong>loxacin resistant strains from bovines. It has also been shown that in<br />
vitro passages <strong>of</strong> mycoplasmas in the presence <strong>of</strong> enr<strong>of</strong>loxacin results in the<br />
development <strong>of</strong> fluoroquinolone resistance (Gautier-Bouchardon et al., 2002;<br />
Reinhardt et al., 2002; Bébéar et al., 1997). A possible explanation for the high<br />
prevalence <strong>of</strong> fluoroquinolone resistance in the present study might be the frequent<br />
use <strong>of</strong> enr<strong>of</strong>loxacin to treat E. coli diarrhea in neonatal <strong>and</strong> recently weaned piglets. In<br />
all the herds with a fluoroquinolone resistant M. hyopneumoniae isolate, these<br />
antibiotics were used during the suckling period. On only 5 herds with a fully<br />
susceptible M. hyopneumoniae isolate, fluoroquinolones were sporadically used to<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
treat suckling piglets. The MIC <strong>of</strong> enr<strong>of</strong>loxacine <strong>of</strong> M. hyopneumoniae isolate 20 was<br />
>1. On the originating herd fluoroquinolones were used as well in suckling, nursery<br />
<strong>and</strong> grow-finishing pigs.<br />
The MICs <strong>of</strong> oxytetracycline <strong>and</strong> doxycycline did not show a clear bimodal<br />
frequency distribution range <strong>and</strong> the MICs <strong>of</strong> oxytetracycline were lower than the<br />
breakpoint proposed by Hannan et al. (1997a), indicating no acquired resistance<br />
against these antibiotics although 62 % <strong>of</strong> the herds selected for this study used<br />
tetracycline antibiotics to treat nursery <strong>and</strong> grow-finishing pigs. M. hyopneumoniae<br />
isolates with decreased oxytetracycline susceptibility have been isolated in Japan<br />
(Inamoto et al., 1994). Acquired resistance against chlortetracycline has been<br />
described by Yamamoto <strong>and</strong> Koshimizu (1984), Inamoto et al. (1994) <strong>and</strong> Etheridge<br />
et al. (1979). For tetracycline resistance we used the antibiotics oxytetracycline <strong>and</strong><br />
doxycycline since stability <strong>of</strong> chlortetracycline is rather poor (Ray <strong>and</strong> Newton, 1991)<br />
<strong>and</strong> due to the long incubation period necessary, one cannot be sure <strong>of</strong> the activity<br />
after this prolonged period <strong>of</strong> incubation.<br />
CONCLUSION<br />
This study is the first description <strong>of</strong> acquired resistance in M. hyopneumoniae<br />
field isolates to macrolides, lincosamides <strong>and</strong> fluoroquinolones. Resistance against<br />
other antimicrobials was not detected confirming that antimicrobial resistance does<br />
not yet pose a major problem for treatment <strong>of</strong> M. hyopneumoniae infections (Hannan<br />
et al., 1989; ter Laak et al., 1991; Inamoto et al., 1994; Hannan et al., 1997a,b).<br />
However, the rather high frequency <strong>of</strong> fluoroquinolone resistance is worrying <strong>and</strong><br />
warrants prudent use <strong>of</strong> these antibiotics.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by the Federal Public Service <strong>of</strong> Public <strong>Health</strong>, Food<br />
Chain Security <strong>and</strong> Environment, Brussels, Belgium, grant no. S-6039.<br />
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2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
REFERENCES<br />
1. Aarestrup, F.M. & Friis, N.F. (1998). Antimicrobial susceptibility testing <strong>of</strong> Mycoplasma<br />
hyosynoviae isolated from pigs during 1968-1971 <strong>and</strong> during 1995 <strong>and</strong> 1996. Veterinary<br />
Microbiology 61, 33-39.<br />
2. Bébéar, C. & Bébéar, C. (2002). Antimycoplasmal agents, p.545-566. In S. Razin <strong>and</strong> R.<br />
Herrmann (ed.), Molecular biology <strong>and</strong> pathogenicity <strong>of</strong> mycoplasmas. Kluwer Academic /<br />
Plenum publishers, New York.<br />
3. Bébéar, C.M., Bové, J. M., Bébéar, C. & Renaudin, J. (1997). Characterization <strong>of</strong><br />
Mycoplasma hominis mutations involved in resistance to fluoroquinolones. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 41, 269-273.<br />
4. Bébéar, C.M., Renaudin, J., Charron, A., Renaudin, H., De Barbeyrac, B., Schaeverbeke,<br />
T. & Bébéar, C. (1999). Mutations in the gyrA, parC <strong>and</strong> parE genes associated with<br />
fluoroquinolone resistance in clinical isolates <strong>of</strong> Mycoplasma hominis. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 43, 954-956.<br />
5. Butaye, P. (2000). Antimicrobial resistance in Enterococcus faecium <strong>and</strong> Enterococcus faecalis<br />
strains from animals <strong>and</strong> animal-derived foods. Ph.D. thesis. Ghent University, Belgium.<br />
6. Butaye, P., Devriese, L.A. & Haesebrouck, F. (2003). Antimicrobial growth promotors used in<br />
animal feed: effects <strong>of</strong> less well known antibiotics on gram-positive bacteria. Clinical<br />
Microbiology Re<strong>view</strong>s 16, 175-188.<br />
7. Caron, J., Ouardani, M. & Dea, S. (2000). Diagnosis <strong>and</strong> differentiation <strong>of</strong> Mycoplasma<br />
hyopneumoniae <strong>and</strong> Mycoplasma hyorhinis infections in pigs by PCR amplification <strong>of</strong> the p36<br />
<strong>and</strong> p46 genes. Journal <strong>of</strong> Clinical Microbiology 38, 1390-1396.<br />
8. Etheridge, J.R., Lloyd, L.C. & Cottew, G.S. (1979). Resistance <strong>of</strong> Mycoplasma<br />
hyopneumoniae to chlortetracycline. Australian Veterinary Journal 55, 40.<br />
9. Friis, N.F. (1971ª). A selective medium for Mycoplasma suipneumoniae. Acta Veterinaria<br />
Sc<strong>and</strong>inavia 12, 454-456.<br />
10. Friis, N.F. (1971b). Mycoplasmas cultivated from the respiratory tract <strong>of</strong> Danish pigs. Acta<br />
Veterinaria Sc<strong>and</strong>inavia 12, 69-79.<br />
11. Friis, N.F. (1975). Some recommendations concerning primary isolation <strong>of</strong> Mycoplasma<br />
suipneumoniae <strong>and</strong> Mycoplasma flocculare. Nordisk Veterinaer Medicin 27, 337-339.<br />
12. Friis, N.F. (1979). Selective isolation <strong>of</strong> slowly growing acidifying mycoplasmas from swine<br />
<strong>and</strong> cattle. Acta Veterinaria Sc<strong>and</strong>inavia 20, 607-609.<br />
13. Friis, N.F. & Szancer, J. (1994). Sensitivity <strong>of</strong> certain porcine <strong>and</strong> bovine mycoplasmas to<br />
antimicrobial agents in a liquid medium test compared to a disc assay. Acta Veterinaria<br />
Sc<strong>and</strong>inavia 35, 389-394.<br />
14. Gautier-Bouchardon, A.V., Reinhardt, A.K., Kobisch, M. & Kempf, I. (2002). In vitro<br />
development <strong>of</strong> resistance to enr<strong>of</strong>loxacin, erythromycin, tylosin, tiamulin <strong>and</strong> oxytetracycline in<br />
Mycoplasma gallisepticum, Mycoplasma iowae <strong>and</strong> Mycoplasma synoviae. Veterinary<br />
Microbiology 88, 47-58.<br />
15. Hannan, P.C.T. (2000). Guidelines <strong>and</strong> recommendations for antimicrobial minimum inhibitory<br />
concentration (MIC) testing against veterinary mycoplasma species. Veterinary Research 31,<br />
373-395.<br />
- 121 -
2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
16. Hannan, P.C.T., O’Hanlon, P.J. & Rogers, N.H. (1989). In vitro evaluation <strong>of</strong> various<br />
quinolone antibacterial agents against veterinary mycoplasmas <strong>and</strong> porcine respiratory bacterial<br />
pathogens. Research in Veterinary Science 46, 202-211.<br />
17. Hannan, P.C.T., Windsor, H.M., de Jong A., Schmeer, N. & Stegemann, M. (1997a).<br />
Comparitive susceptibilities <strong>of</strong> various animal-pathogenic mycoplasmas to fluoroquinolones.<br />
Antmicrobial Agents <strong>and</strong> Chemotherapy 41, 2037-2040.<br />
18. Hannan, P.C.T., Windsor, H.M. & Ripley, P.H. (1997b). In vitro susceptibilities <strong>of</strong> recent<br />
field isolates <strong>of</strong> Mycoplasma hyopneumoniae <strong>and</strong> Mycoplasma hyosynoviae to valnemulin<br />
(Econor), tiamulin <strong>and</strong> enr<strong>of</strong>loxacin <strong>and</strong> the in vitro development <strong>of</strong> resistance to certain<br />
antimicrobial agents in Mycoplasma hyopneumoniae. Research in Veterinary Science 36, 157-<br />
160.<br />
19. Inamoto, T., Takahashi, K., Yamamoto, K., Nakai, Y. & Ogimoto, K. (1994). Antibiotic<br />
susceptibility <strong>of</strong> Mycoplasma hyopneumoniae isolated from swine. The Journal <strong>of</strong> Veterinary<br />
Medical Science 56, 393-394.<br />
20. Kobayashi, H., Morozumi, T., Munthali, G., Mitani, K., Ito, N. & Yamamoto, D. (1996a).<br />
Macrolide susceptibility <strong>of</strong> Mycoplasma hyorhinis isolated from pigs. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 40, 1030-1032.<br />
21. Kobayashi, H., Sonmez, N., Morozumi, T., Mitani, K., Ito, N., Shiono, H. & Yamamoto, K.<br />
(1996b). In vitro susceptibility <strong>of</strong> Mycoplasma hyosynoviae <strong>and</strong> M. hyorhinis to antimicrobial<br />
agents. The Journal <strong>of</strong> Veterinary Medical Science 58, 1107-1111.<br />
22. Léon, F.A., Madec, F., Taylor, N.M. & Kobisch M. (2001). Seroepidemiology <strong>of</strong> Mycoplasma<br />
hyopneumoniae in pigs from farrow-to-finish farms. Veterinary Microbiology 78, 331-341.<br />
23. Levisohn, S. (1981). Antibiotic sensitivity patterns in field isolates <strong>of</strong> Mycoplasma<br />
gallisepticum as a guide to chemotherapy. Israel Journal <strong>of</strong> Medical Sciences 17, 661-666.<br />
24. Lucier, T.S., Heitzman, K., Liu, S-K. & Hu, P-C. (1995). Transition mutations in the 23S<br />
rRNA <strong>of</strong> erythromycin resistant isolates <strong>of</strong> Mycoplasma pneumoniae. Antmicrobial Agents <strong>and</strong><br />
Chemotherapy 39, 2770-2773.<br />
25. Mattsson, J.G., Bergström, K., Wallgren, P. & Johansson, K.E. (1995). Detection <strong>of</strong><br />
Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification <strong>of</strong> the 16s RNA<br />
gene. Journal <strong>of</strong> Clinical Microbiology 33, 893-897.<br />
26. Ray, A. & Newton, V. (1991). Use <strong>of</strong> high-performance liquid chromatography to monitor<br />
stability <strong>of</strong> tetracycline <strong>and</strong> chlortetracycline in susceptibility determinations. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 35, 1264-1266.<br />
27. Reinhardt, A.K., Bébéar, C.M., Kobisch, M., Kempf, I. & Gautier-Bouchardon, A.V.<br />
(2002). Characterization <strong>of</strong> mutations in DNA Gyrase <strong>and</strong> Topoisomerase IV involved in<br />
quinolone resistance <strong>of</strong> Mycoplasma gallisepticum mutants obtained in vitro. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 46, 590-593.<br />
28. Tanner, A.C. & Wu, C.C. (1992). Adaptation <strong>of</strong> the Sensititre ® broth microdilution technique<br />
to antimicrobial susceptibility testing <strong>of</strong> Mycoplasma gallisepticum. Avian Diseases 36, 714-<br />
717.<br />
29. Tanner, A.C., Erickson, B.Z. & Ross, R.F. (1993). Adaptation <strong>of</strong> the Sensititre ® broth<br />
microdilution technique to antimicrobial susceptibility testing <strong>of</strong> Mycoplasma hyopneumoniae.<br />
Veterinary Microbiology 36, 301-306.<br />
30. Ter Laak, E.A., Noordergraaf, J.H. & Verschure, M.H. (1993). Susceptibilities <strong>of</strong><br />
Mycoplasma bovis, Mycoplasma dispar <strong>and</strong> Ureaplasma diversum strains to antimicrobial<br />
agents in vitro. Antimicrobial Agents <strong>and</strong> Chemotherapy 37, 317-321.<br />
- 122 -
2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES<br />
31. Ter Laak, E.A., Pijpers, A., Noordergraaf, J.H., Schroevers, E.C. & Verheijden, J.H.M.<br />
(1991). Comparison <strong>of</strong> methods for in vitro testing <strong>of</strong> susceptibility <strong>of</strong> porcine mycoplasma<br />
species to antimicrobial agents. Antimicrobial Agents <strong>and</strong> Chemotherapy 35, 228-233.<br />
32. Thomas, A., Nicolas, C., Dizier, I., Mainil, J. & Linden, A. (2003). Antibiotic susceptibilities<br />
<strong>of</strong> recent Belgian Mycoplasma bovis isolates. The Veterinary Record 153, 428-431.<br />
33. Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F. & de Kruif, A. (2002).<br />
Patterns <strong>of</strong> Mycoplasma hyopneumoniae infections in Belgian farrow-to-fiinish pig herds with<br />
diverging disease-course. Journal <strong>of</strong> Veterinary Medicine, Series B 49, 349-353.<br />
34. Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. & Haesebrouck, F.<br />
(2003). Evaluation <strong>of</strong> virulence <strong>of</strong> Mycoplasma hyopneumoniae field isolates. Veterinary<br />
Microbiology 97, 177-190.<br />
35. Weisblum, B. (1995). Insights into erythromycin action from studies <strong>of</strong> its activity as inducer <strong>of</strong><br />
resistance. Antmicrobial Agents <strong>and</strong> Chemotherapy 39, 797-805.<br />
36. Yamamoto, K. & Koshimizu, K. (1984). In vitro susceptibility <strong>of</strong> Mycoplasma hyopneumoniae<br />
to antibiotics, p. 116. Proceedings <strong>of</strong> the 8 th International Pig Veterinary Society Congress,<br />
Ghent, Belgium.<br />
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2.2.2 CHARACTERIZATION OF IN VIVO ACQUIRED<br />
RESISTANCE OF M. HYOPNEUMONIAE TO MACROLIDES<br />
AND LINCOSAMIDES<br />
Modified from:<br />
CHARACTERISATION OF IN VIVO ACQUIRED RESISTANCE OF MYCOPLASMA HYOPNEUMONIAE TO<br />
MACROLIDES AND LINCOSAMIDES<br />
T. STAKENBORG, J. VICCA, P. BUTAYE, D. MAES, F.C. MINION, J. PEETERS, A. DE KRUIF & F.<br />
HAESEBROUCK<br />
MICROBIAL DRUG RESISTANCE 2005, 11, 291-295<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
SUMMARY<br />
Macrolides <strong>and</strong> related antibiotics are used to control mycoplasma infections in<br />
the pig industry worldwide. Some porcine mycoplasmas, however, survive these<br />
treatments by acquiring resistance. The mechanism <strong>of</strong> acquired resistance to<br />
macrolides <strong>and</strong> lincosamides was studied in more detail for Mycoplasma<br />
hyopneumoniae (M. hyopneumoniae) by comparing both the phenotype <strong>and</strong> genotype<br />
<strong>of</strong> a resistant field isolate to 5 susceptible isolates. The minimal inhibitory<br />
concentrations (MICs) were significantly higher for the resistant isolate for all<br />
antibiotics tested. The MICs for the 16-membered macrolide tylosin ranged from 8 to<br />
16 µg for the resistant isolate <strong>and</strong> from 0.03 to 0.125 µg/ml for the 5 susceptible<br />
isolates. The MICs for the 15-membered macrolides <strong>and</strong> lincosamides were higher<br />
than 64 µg/ml for the resistant isolate while only 0.06 to 0.5 µg/ml for the susceptible<br />
isolates. M. hyopneumoniae isolates are intrinsically resistant to the 14-membered<br />
macrolides due to a G2057A transition (E. coli numbering) in their 23S rDNA.<br />
Therefore, high MICs were observed for all isolates, although the MICs for the<br />
resistant isolate were clearly increased. An additional, acquired A2058G point<br />
mutation was found in the 23S rRNA gene <strong>of</strong> the resistant isolate. No differences<br />
linked to resistance were found in the ribosomal proteins L4 <strong>and</strong> L22. The present<br />
study showed that 23S rRNA mutations resulting in resistance to macrolides <strong>and</strong><br />
lincosamides as described in other Mycoplasma spp. also occur under field conditions<br />
in M. hyopneumoniae.<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
INTRODUCTION<br />
Mycoplasmas are the smallest free-living organisms, carrying no cell wall. As<br />
a consequence, they are naturally resistant to antibiotics interfering with cell wall<br />
synthesis. Additionally, a number <strong>of</strong> reports indicate a decrease in susceptibility <strong>of</strong><br />
mycoplasmas against widely used antimicrobial agents, including the macrolides,<br />
lincosamides <strong>and</strong> streptogramins (MLS) (Hannan et al., 1997; Aarestrup <strong>and</strong> Friis,<br />
1998; Furneri et al., 2001; Okazaki et al., 2001; Thomas et al., 2003; Vicca et al.,<br />
2004). MLS antibiotics have overlapping binding sites on the 23S rRNA <strong>and</strong>, hence,<br />
related antimicrobial activities. By binding to domain V <strong>of</strong> the 23S rRNA, they inhibit<br />
protein synthesis by means <strong>of</strong> blocking the path through which nascent peptides exit<br />
the ribosome (Retsema <strong>and</strong> Fu, 2001; Tenson et al., 2003). Additional studies have<br />
mapped the recognition site <strong>of</strong> the 14-membered macrolide erythromycin <strong>and</strong> its<br />
derived ketolides to domain II <strong>and</strong> IV <strong>of</strong> the 23S rRNA as well (Vester <strong>and</strong><br />
Douthwaite, 2001; Berisio et al., 2003; Schlünzen et al., 2003).<br />
Bacterial species containing only 1 or 2 copies <strong>of</strong> rRNA genes, like all<br />
Mycoplasma species, tend to use mutations at bases 2057-2059 <strong>of</strong> the 23S rRNA as a<br />
way <strong>of</strong> acquiring resistance (Furneri et al., 2000; Hansen et al., 2002). Some<br />
mycoplasmas, like M. hyopneumoniae, are intrinsically resistant to 14-membered<br />
macrolides due to a G2057A transition in their 23S rDNA (Stemke et al., 1994;<br />
Ludwig et al., 1992; Furneri et al., 2000). Additional resistance to MLS antibiotics<br />
due to mutations at position 2609-2611 has been observed for several bacterial species<br />
(Garza-Ramos et al., 2001; Canu et al., 2002; Pereyre et al., 2002). A mutation at<br />
position 2062 was linked to resistance against josamycin, a 16-membered macrolide,<br />
in an in vitro selected M. hominis strain (Furneri et al., 2001; Hansen et al., 2002). For<br />
a number <strong>of</strong> other bacteria, mutations in the L4 <strong>and</strong> L22 binding proteins were linked<br />
to MLS resistance (Tait-Kamradt et al., 2000; Canu et al., 2002; Pihlajamaki et al.,<br />
2002; Jones et al., 2003; Schlünzen et al., 2003; Farrell et al., 2004; Franceschi et al.,<br />
2004; Pereyre et al., 2004). Nonetheless, acquired resistance to MLS in Mycoplasma<br />
species is rarely documented or is induced in vitro rather than observed in field<br />
isolates. The aim <strong>of</strong> this study was to fully characterize both phenotypically <strong>and</strong><br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
genetically the in vivo acquired resistance to macrolides <strong>and</strong> lincosamides in a M.<br />
hyopneumoniae field isolate.<br />
MATERIALS AND METHODS<br />
Media, bacterial isolates <strong>and</strong> antibiotics<br />
The F2, F5, F6, F18 <strong>and</strong> F19 isolates were all Belgian M. hyopneumoniae<br />
field-isolates obtained from lung tissue <strong>of</strong> fattening pigs collected at slaughterhouses.<br />
The J-strain (ATCC 25934) was used as a control strain. The Friis’ broth (Friis, 1975)<br />
without added antibiotics was used to grow the M. hyopneumoniae isolates.<br />
Erythromycin, tylosin <strong>and</strong> clindamycin were obtained from Sigma (UK). The<br />
other antibiotics were obtained from the drug's manufacturer as reference powder:<br />
lincomycin <strong>and</strong> azithromycin were kindly supplied by Pfizer (NY, USA),<br />
clarithromycin by Abott (Il, USA).<br />
The Minimal inhibitory concentration (MIC) test was performed using a<br />
macro broth dilution technique. Antibiotic stock solutions <strong>of</strong> 1,000 µg/ml were freshly<br />
prepared the day <strong>of</strong> use according to guidelines described by the National Committee<br />
for Clinical Laboratory St<strong>and</strong>ards (NCCLS, 1999). Clarithromycin, erythromycin <strong>and</strong><br />
azithromycin were dissolved in a minimum amount <strong>of</strong> ethanol <strong>and</strong> further diluted in<br />
water. All other antibiotics were directly dissolved in water. For each antimicrobial,<br />
two-fold dilutions ranging from 0.015 µg/ml to 64 µg/ml were prepared <strong>and</strong> each<br />
strain was tested twice. Broths were inoculated with the M. hyopneumoniae isolates,<br />
resulting in approximately 10 5 colour-changing units (CCU) per ml in a final volume<br />
<strong>of</strong> 2 ml. An additional tube contained only medium <strong>and</strong> was used as a negative<br />
control, while a second tube without antibiotics served as a positive control. The MIC<br />
was defined as the lowest concentration <strong>of</strong> antimicrobial agent in which no growth <strong>of</strong><br />
M. hyopneumoniae was observed. Growth was detected by a colour change <strong>of</strong> the<br />
phenol red indicator from red to yellow due to glucose fermentation. The test was<br />
ended when no more colour change in the tubes was visible during two consecutive<br />
days.<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
DNA sequencing <strong>of</strong> the L4- <strong>and</strong> L22-genes <strong>and</strong> domain V <strong>of</strong> the 23S rDNA<br />
To sequence the L4 <strong>and</strong> L22 proteins, primers were designed based on the<br />
M. hyopneumoniae genome sequence <strong>of</strong> reference strain 232 (Minion et al., 2004).<br />
Primers for the amplification <strong>of</strong> domain V <strong>of</strong> the 23S rDNA were selected based on<br />
the M. hyopneumoniae ATCC 27719 strain (Genbank accession number X68421). All<br />
primers <strong>and</strong> reaction conditions used are listed in Table 1. After purification <strong>of</strong> the<br />
PCR product on Microcon 100 columns (Millipore, MA, USA), both str<strong>and</strong>s were<br />
sequenced on a CEQ8000 sequencer (Beckmann, UK) according to the<br />
manufacturer’s instructions. The sequences were aligned for further analysis using<br />
Clustal W (V1.82). The sequences <strong>of</strong> domain V <strong>of</strong> the 23S rDNA <strong>of</strong> the M.<br />
hyopneumoniae isolates were compared with sequences <strong>of</strong> E. coli <strong>and</strong> other<br />
Mycoplasma species extracted from Genbank (Figure 1).<br />
Table 1: Selected primers used for the amplification <strong>and</strong> sequencing <strong>of</strong> the 23S, L4<br />
<strong>and</strong> L22 genes <strong>of</strong> the M. hyopneumoniae J-reference <strong>and</strong> field strains.<br />
Primer name Primer Sequence (5’ → 3’) Number <strong>of</strong> cycles (Cycle conditions) 1<br />
L4 FOR AGCATTCAAAGTCAGAAAAC 25 (30” 94°C; 30” 47.6°C; <strong>and</strong> 1’ 72°C)<br />
L4 REV<br />
GATTCTCTTCTCCAAATTAG<br />
L22 FOR AGCAGTCGCTTCACTCAAAA 25 (30” 94°C; 30” 52.5°C; <strong>and</strong> 1’ 72°C)<br />
L22 REV<br />
ACCTCTTTTTCTTGCGCTAA<br />
23S FOR GGTAGCGAAATTCCTTGTCA 25 (30” 94°C; 30” 55.2°C; <strong>and</strong> 1’ 72°C)<br />
23S REV<br />
GAGCAGCTCTCATCAATATTCC<br />
1 : All PCRs, unless stated otherwise, were performed using 2U <strong>of</strong> recombinant Taq polymerase, 5 µl <strong>of</strong><br />
10X PCR buffer, 75 nmol MgCl2, 10 nmol <strong>of</strong> each dNTP <strong>and</strong> 10 pmol <strong>of</strong> both forward <strong>and</strong> reverse<br />
primer.<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
|2040 |2050 |2060 |2070 |2080 |2090 |2100<br />
E. coli a GTACCCGCGG CAAGACGGAA AGACCCCGTG AACCTTTACT ATAGCTTGAC ACTGAACATT GAGCCTTGATG<br />
M. hyopneumoniae b TTACCCGCAT CAAGACGAAA AGACCCCGTG GAGCTTTACT ATAACTTCGT ATTGAGAATT GGTTTATTATG<br />
M. hyopneumoniae F19 TTACCCGCAT CAAGACGAGA AGACCCCGTG GAGCTTTACT ATAACTTCGT ATTGAGAATT GGTTTATTATG<br />
M. hominis AGACCCGCAT CTAGACGAAA AGACCCCGTG GAGCTTTACT ATAACTTCAT ATTGGAGTTT GATTTAACATG<br />
M. fermentans GTACCCGCAT CAAGACGAAA AGACCCCATG GAGCTTTACT ACAGTTTCGT ATTGGAACTT GGTCTAACATG<br />
M. genitalium TTAGGCGCAA CGGGACGGAA AGACCCCGTG AAGCTTTACT GTAGCTTAAT ATTGATCAAA ACACCACCATG<br />
M. pneumoniae TTAGGCGCAA CGGGACGGAA AGACCCCGTG AAGCTTTACT GTAGCTTAAT ATTGATCAGG ACATTATCATG<br />
M. gallisepticum TTAGGCGCAA CGGGACGGAA AGACCCCATG AAGCTTTACT GTAACTTAAT ATTGGGCAGA GTTTAGACATA<br />
M. penetrans TTAGGTGCGG TTAGACAAAA AGACCCCATG AAGCTTTACT GTAGCTTAAT ATTGGAAAAA TTTATTTCATT<br />
M. flocculare TTACCCGCAT CAAGACGAAA AGACCCCGTG GACGTTTACT ATAACTTCGT ATTGAGAATT GGTTTATTATG<br />
M. hyorhinis CGAACCGTAG TACGCTAAAA AGTGCCCGGA TGACTTGTGG ATAGC----- GGTGAAATTC CAATCGA-ACC<br />
M. pulmonis c CGAACCGTAG TACGCTGAAA AGTGCCCGGA TGACTTGTGA ATAGC----- GGTGAAATTC CAATCGA-ACC<br />
* * * * ** *** ** * ** *<br />
a<br />
Sequences were extracted from Genbank. Accession numbers are: Escherichia coli, ECO278710; M.<br />
hominis, AF443617; M. fermentans, AF422142; M. genitalium, U39634; M. pneumoniae, AE000007;<br />
M. gallisepticum, AE016968; M. penetrans, AP004174; M. flocculare, MYC16SR; M. hyorhinis,<br />
AF121891; M. pulmonis, AL445565.<br />
b<br />
No differences in the 23S DNA <strong>of</strong> isolate F2, F5, F6, F18, F23 <strong>and</strong> J were observed.<br />
c<br />
Resistance due to A2062 point mutations have been described elsewhere 10<br />
Figure 1: Multiple sequence-alignment <strong>of</strong> the 23S rDNA <strong>of</strong> different Mycoplasma<br />
spp. compared to E. coli. Nucleotides related to resistance to macrolides are presented<br />
in bold. The G2058A transition <strong>of</strong> the resistant M. hyopneumoniae F19 isolate is<br />
underlined as well.<br />
RESULTS<br />
Because the isolates grew at different rates, not all MICs were read on the<br />
same day. The J-reference strain grew faster than the other isolates, <strong>and</strong> the MICs <strong>of</strong><br />
the antibiotics were reached after approximately 5 days, while for the other isolates it<br />
took up to one week.<br />
The MICs for the isolates are presented in Table 2. For each isolate, the MIC<br />
values <strong>of</strong> the replicates were equal to or differed from each other by only one dilution.<br />
The MICs for erythromycin <strong>and</strong> clarithromycin ranged from 8 – 32 µg/ml for all<br />
isolates tested, except for the resistant F19 isolate. For this isolate, the MIC values<br />
exceeded the highest concentration (64 µg/ml) tested. All isolates, except the F19<br />
isolate, were also susceptible to the other antibiotics tested. The MICs ranged from<br />
0.06 – 0.25 µg/ml for the 15-membered macrolide, azithromycin; from 0.03 – 0.125<br />
µg/ml for the 16-membered macrolide, tylosin; <strong>and</strong> from 0.125 – 0.5 µg/ml for the<br />
lincosamides, lincomycin <strong>and</strong> clindamycin. The MICs for the resistant isolate (F19)<br />
also exceeded the highest concentration <strong>of</strong> 64 µg/ml for these antibiotics, with the<br />
exception <strong>of</strong> tylosin (MICs ranging from 8-16 µg/ml).<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
Sequence analysis <strong>of</strong> the 23S rDNA as shown in Figure 1, demonstrates the<br />
intrinsic resistance <strong>of</strong> certain Mycoplasma spp. to 14-membered macrolides due to a<br />
G2057A transition. An acquired A2058G transition was found exclusively in the<br />
resistant M. hyopneumoniae isolate. Only a small number <strong>of</strong> differences were present<br />
in the L22 proteins (>97% identity at DNA level). Protein L4 proved very conserved<br />
(>99% identical at DNA level) since no amino acid substitutions were found between<br />
any <strong>of</strong> the isolates (data not shown).<br />
Table 2: MICs (µg/ml) <strong>of</strong> MLS-antimicrobial agents for Belgian M. hyopneumoniae<br />
field isolates <strong>and</strong> the M. hyopneumoniae J reference strain obtained by the broth<br />
dilution test carried out in tw<strong>of</strong>old.<br />
Isolate<br />
MIC (µg/ml)<br />
Azithromycin Tylosin Erythromycin Clarithromycin Clindamycin Lincomycin<br />
F2 0.125 a 0.03125 8 16 0.0625 0.25<br />
F5 0.125-0.25 0.03125 8 16 0.125-0.25 0.25<br />
F6 0.0625-0.125 0.03125 16 32 0.125 0.125<br />
F18 0.0625 0.0625 32 32 0.125-0.25 0.125<br />
F19 >64 8-16 >64 >64 >64 >64<br />
J 0.25 0.125 32 32 0.5 0.5<br />
a one value means that no difference between the repeated tests was observed.<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
DISCUSSION<br />
Since M. hyopneumoniae isolates are difficult to grow on agar plates <strong>and</strong><br />
colonies are hard to detect, a serial broth dilution technique using Friis’ medium was<br />
chosen to test for antibiotic resistance. This technique, based on an earlier report <strong>of</strong><br />
Ter Laak et al. (1991), was performed in duplicate <strong>and</strong> proved to be reproducible.<br />
The J-reference strain seemed better adapted to the Friis’ medium <strong>and</strong> grew<br />
faster than the other isolates. This may explain the higher MICs, up to one dilution,<br />
for this strain. Nevertheless, all individual MICs were very similar for the susceptible<br />
isolates <strong>and</strong> clearly differed from those <strong>of</strong> the resistant F19 isolate. Moreover, MICs<br />
for the sensitive isolates were consistent with previous reports (Ter Laak et al., 1991;<br />
Tanner et al., 1993; Inamoto et al., 1994).<br />
Although M. hyopneumoniae isolates are naturally resistant to 14-membered<br />
macrolides due to a G2057A transition <strong>of</strong> the 23S gene (Vester <strong>and</strong> Douthwaite,<br />
2001), even higher MICs were observed for the resistant isolate. This increased<br />
resistance may well be explained by the observed A2058G transition since<br />
footprinting patterns examining the drug binding sites in other bacteria identified<br />
these specific nucleotides (Hansen et al., 1999; Edelstein, 2004). Hence, a lower<br />
affinity <strong>of</strong> the drugs to the 23S rRNA, due to the acquired mutation, results in a<br />
decreased antimicrobial activity. Apart from this increased insensitivity to<br />
erythromycin <strong>and</strong> clarithromycin, the F19 field isolate proved also resistant to all<br />
other antibiotics tested. This is in agreement with earlier reports in other bacterial<br />
species where mutations at position 2058 lead to high MLS resistant isolates (Vester<br />
<strong>and</strong> Douthwaite, 2001; Edelstein, 2004). This A2058G transition may even be the<br />
most frequently observed substitution in vivo in association with MLS resistance<br />
(Vester <strong>and</strong> Douthwaite, 2001) <strong>and</strong> was also found for clinical isolates <strong>of</strong><br />
M. pneumoniae (Okazaki et al., 2001), although only limited data exist for in vivo<br />
acquired MLS resistance in Mycoplasma species.<br />
Recently, resistance to macrolides <strong>and</strong> lincosamides has been linked to<br />
modifications in the L4 <strong>and</strong> L22 rRNA-binding proteins (Gabashvili et al., 2001;<br />
O’Connor et al., 2004), especially in pneumococcal strains (Tait-Kamradt et al., 2000;<br />
Gabashvili et al., 2001; Canu et al., 2002; Pihlajamaki et al., 2002; Jones et al., 2003;<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
Franceschi et al., 2004; O’Connor et al., 2004). In the present studies, none <strong>of</strong> the<br />
variations in these proteins were uniquely present in the resistant M. hyopneumoniae<br />
field isolate F19, <strong>and</strong> it is therefore unlikely that they are associated with acquired<br />
resistance. This is in agreement with an earlier report <strong>of</strong> resistant clinical strains <strong>of</strong><br />
M. hominis <strong>and</strong> M. fermentans (Pereyre et al., 2002).<br />
In in vitro experiments, resistance to tylosin was obtained in M. hyopneumoniae<br />
isolates after only 7 or fewer passages in selective media (Hannan et al., 1997),<br />
indicating that acquired resistance due to mutations <strong>of</strong> the 23S RNA may occur quite<br />
fast for bacteria like M. hyopneumoniae, which only possess one copy <strong>of</strong> the rRNA<br />
operon (Stemke et al., 1994). The relation between the use <strong>of</strong> macrolides in pig<br />
rearing <strong>and</strong> the occurrence <strong>of</strong> acquired resistance has been indirectly demonstrated for<br />
M. hyosynoviae <strong>and</strong> other porcine bacteria (Aarestrup <strong>and</strong> Carstensen, 1998;<br />
Aarestrup <strong>and</strong> Friis, 1998; Butaye et al., 2001). In contrast, the prevalence <strong>of</strong> acquired<br />
resistance to macrolides for M. hyopneumoniae in the field is most likely low<br />
(Inamoto et al., 1994; Vicca et al., 2004). It is possible that resistant isolates do not<br />
spread easily <strong>and</strong> that occurrence <strong>of</strong> resistance remains localized in an area or even<br />
within a herd. Indeed, earlier RAPD data (Artiushin <strong>and</strong> Minion, 1996) <strong>and</strong> our own<br />
PFGE results (Stakenborg et al., 2005) show an enormous diversity between isolates<br />
from different farms, suggesting that clones do not readily spread in the environment,<br />
although further research on this issue is needed. Since the type <strong>of</strong> resistance<br />
described above is not encoded on a mobile element, <strong>and</strong> therefore not transferable<br />
between different isolates, the genetic stability <strong>of</strong> the mutation may also be an<br />
important factor. Although the A2058G transition has distinct advantages over the<br />
wild-type in the presence <strong>of</strong> macrolides, the situation may be different in the absence<br />
<strong>of</strong> the drugs (Vester <strong>and</strong> Douthwaite, 2001). In any event, the emergence <strong>of</strong> this<br />
resistance asks for a continuous monitoring, as it may have important therapeutic<br />
implications in the treatment <strong>of</strong> mycoplasma infections.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by a grant <strong>of</strong> the Federal Agency <strong>of</strong> <strong>Health</strong>, Food<br />
Chain Security <strong>and</strong> Environment (Grant number S-6136).<br />
The authors thank Sara Tistaert for skilful technical assistance.<br />
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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
REFERENCES<br />
1 Aarestrup, FM. & Carstensen, B. (1998). Effect <strong>of</strong> tylosin used as a growth promoter on the<br />
occurrence <strong>of</strong> macrolide-resistant enterococci <strong>and</strong> staphylococci in pigs. Microbial Drug<br />
Resistance 4, 307-312.<br />
2 Aarestrup, FM. & Friis, N.F. (1998). Antimicrobial susceptibility testing <strong>of</strong> Mycoplasma<br />
hyosynoviae isolated from pigs during 1968 to 1971 <strong>and</strong> during 1995 <strong>and</strong> 1996. Veterinary<br />
Microbiology 61, 33-39.<br />
3 Artiushin, S. & Minion, F.C. (1996). Arbitrarily primed PCR analysis <strong>of</strong> Mycoplasma<br />
hyopneumoniae field isolates demonstrates genetic heterogeneity. International Journal <strong>of</strong><br />
Systemic Bacteriology 46, 324-328.<br />
4 Berisio, R., Harms, J., Schluenzen, F., Zarivach, R., Hansen, HA., Fucini, P. & Yonath, A.<br />
(2003). Structural insight into the antibiotic action <strong>of</strong> telithromycin against resistant mutants.<br />
Journal <strong>of</strong> Bacteriology 185, 4276-4279.<br />
5 Butaye, P., Devriese, LA. & Haesebrouck, F. (2001). Differences in antibiotic resistance<br />
patterns <strong>of</strong> Enterococcus faecalis <strong>and</strong> Enterococcus faecium strains isolated from farm <strong>and</strong> pet<br />
animals. Antimicrobial Agents <strong>and</strong> Chemotherapy 45, 1374-1378.<br />
6 Canu, A., Malbruny, B., Coquemont, M., Davies, T.A., Appelbaum, P.C. & Leclercq, R.<br />
(2002). Diversity <strong>of</strong> ribosomal mutations conferring resistance to macrolides, clindamycin,<br />
streptogramin, <strong>and</strong> telithromycin in Streptococcus pneumoniae. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 46, 125-131.<br />
7 Edelstein, PH. (2004). Pneumococcal resistance to macrolides, lincosamides, ketolides, <strong>and</strong><br />
streptogramin B agents: molecular mechanisms <strong>and</strong> resistance phenotypes. Clinical Infectious<br />
Diseases (Suppl). 38, S322-327.<br />
8. Farrell, D.J., Morrissey, I., Bakker, S., Buckridge, S., & Felmingham, D. (2004). In vitro<br />
activities <strong>of</strong> telithromycin, linezolid, <strong>and</strong> quinupristin-dalfopristin against Streptococcus<br />
pneumoniae with macrolide resistance due to ribosomal mutations. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 48, 3169-3171.<br />
9. Franceschi, F., Kanyo, Z., Sherer, E.C. & Sutcliffe, J. (2004). Macrolide resistance from the<br />
ribosome perspective. Current Drug Targets - Infectious Disorders 4, 177-91.<br />
10. Friis, NF. (1975). Some recommendations concerning primary isolation <strong>of</strong> Mycoplasma<br />
suipneumoniae <strong>and</strong> Mycoplasma flocculare a survey. Nordisk Veterinaer Medicin 27, 337-339.<br />
11. Furneri, PM., Rappazzo, G., Musumarra, M.P., Di Pietro, P., Catania, L.S. & Roccasalva,<br />
L.S. (2001). Two new point mutations at A2062 associated with resistance to 16-membered<br />
macrolide antibiotics in mutant strains <strong>of</strong> Mycoplasma hominis. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 45, 2958-2960.<br />
12. Furneri, PM., Rappazzo, G., Musumarra, M.P., Tempera, G. & Roccasalva, L.S. (2000).<br />
Genetic basis <strong>of</strong> natural resistance to erythromycin in Mycoplasma hominis. Journal <strong>of</strong><br />
Antimicrobial Chemotherapy 45, 547-548.<br />
13. Gabashvili, I. S., Gregory, S. T., Valle, M., Grassucci, R., Worbs, M., Wahl, M.C.,<br />
Dahlberg, A.E. & Frank, J. (2001). The polypeptide tunnel system in the ribosome <strong>and</strong> its<br />
gating in erythromycin resistance mutants <strong>of</strong> L4 <strong>and</strong> L22. Molecular Cell 8, 181-8.<br />
- 135 -
2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
14. Garza-Ramos, G., Xiong, L., Zhong, P., & Mankin, A. (2001). Binding site <strong>of</strong> macrolide<br />
antibiotics on the ribosome: new resistance mutation identifies a specific interaction <strong>of</strong> ketolides<br />
with rRNA. Journal <strong>of</strong> Bacteriology 183, 6898-6907.<br />
15. Hannan, PC., Windsor, H.M. & Ripley, P.H. (1997). In vitro susceptibilities <strong>of</strong> recent field<br />
isolates <strong>of</strong> Mycoplasma hyopneumoniae <strong>and</strong> Mycoplasma hyosynoviae to valnemulin (Econor),<br />
tiamulin <strong>and</strong> enr<strong>of</strong>loxacin <strong>and</strong> the in vitro development <strong>of</strong> resistance to certain antimicrobial<br />
agents in Mycoplasma hyopneumoniae. Research in Veterinary Science 63, 157-160.<br />
16. Hansen, JL., Ippolito, J.A., Ban, N., Nissen, P., Moore, P.B. & Steitz, T.A. (2002). The<br />
structures <strong>of</strong> four macrolide antibiotics bound to the large ribosomal subunit. Molecular Cell 10,<br />
117-128.<br />
17. Hansen, LH., Mauvais, P. & Douthwaite, S. (1999). The macrolide-ketolide antibiotic binding<br />
site is formed by structures in domains II <strong>and</strong> V <strong>of</strong> 23S ribosomal RNA. Molecular<br />
Microbiology 31, 623-631.<br />
18. Inamoto, T., Takahashi, H., Yamamoto, K., Nakai, Y. & Ogimoto, K. (1994). Antibiotic<br />
susceptibility <strong>of</strong> Mycoplasma hyopneumoniae isolated from swine. The Journal <strong>of</strong> Veterinary<br />
Medical Science 56, 393-394.<br />
19. Jones, RN., Farrell, D.J. & Morrissey, I. (2003). Quinupristin-dalfopristin resistance in<br />
Streptococcus pneumoniae: novel L22 ribosomal protein mutation in two clinical isolates from<br />
the SENTRY antimicrobial surveillance program. Antimicrobial Agents <strong>and</strong> Chemotherapy 47,<br />
2696-2698.<br />
20. Ludwig, W., G. Kirchh<strong>of</strong>, N. Klgubauer, M. Weizenegger, D. Betzl, M. Ehrmann, C.<br />
Hertel, S. Jilg, R. Tatzel, H. Zitzelsberger, S. Liebl, M. Hochberger, J. Shah, D. Lane, PR.<br />
Wallnöfer, <strong>and</strong> KH. Scheifer. (1992). Complete 23S ribosomal RNA sequences <strong>of</strong> Grampositve<br />
bacteria with a low DNA G+C content. System. Applied Microbiology 15, 487-501.<br />
21. Minion, FC., Lefkowitz, E.J., Madsen, M.L., Cleary, B.J., Swartzell, S.M. & Mahairas,<br />
G.G. (2004). The genome sequence <strong>of</strong> Mycoplasma hyopneumoniae strain 232, the agent <strong>of</strong><br />
swine mycoplasmosis. Journal <strong>of</strong> Bacteriology 186, 7123-7133.<br />
22. NCCLS. (1999). Performance st<strong>and</strong>ards for antimicrobial disk <strong>and</strong> dilution susceptibility tests<br />
for bacteria from animals. Approved st<strong>and</strong>ard M31-A. National Committee for Clinical<br />
Laboratory St<strong>and</strong>ards, Pennsylvania, USA, pp. 1-64.<br />
23. O'Connor, M., Gregory, S.T. & Dahlberg, A.E. (2004). Multiple defects in translation<br />
associated with altered ribosomal protein L4. Nucleic Acids Research 32, 5750-6.<br />
24. Okazaki, N., Narita, M., Yamada, S., Izumikawa, K., Umetsu, M., Kenri, T., Sasaki, Y.,<br />
Arakawa, Y. & Sasaki, T. (2001). Characteristics <strong>of</strong> macrolide-resistant Mycoplasma<br />
pneumoniae strains isolated from patients <strong>and</strong> induced with erythromycin in vitro. Microbiology<br />
<strong>and</strong> Immunology 45, 617-20.<br />
25. Pereyre, S., Gonzalez, P., De Barbeyrac, B., Darnige, A., Renaudin, H., Charron, A.,<br />
Raherison, S., Bébéar, C. & CM. Bébéar, C.M. (2002). Mutations in 23S rRNA account for<br />
intrinsic resistance to macrolides in Mycoplasma hominis <strong>and</strong> Mycoplasma fermentans <strong>and</strong> for<br />
acquired resistance to macrolides in M. hominis. Antimicrobial Agents Chemotherapy 46, 3142-<br />
3150.<br />
26. Pereyre, S., Guyot, C., Renaudin, H., Charron, A., Bébéar, C. & Bébéar, C.M. (2004). In<br />
vitro selection <strong>and</strong> characterization <strong>of</strong> resistance to macrolides <strong>and</strong> related antibiotics in<br />
Mycoplasma pneumoniae. Antimicrobial Agents <strong>and</strong> Chemotherapy 48, 460-465.<br />
27. Pihlajamaki, M., Kataja, J., Seppala, H., Elliot, J., Leinonen, M., Huovinen, P. & Jalava, J.<br />
(2002). Ribosomal mutations in Streptococcus pneumoniae clinical isolates. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 46, 654-658.<br />
- 136 -
2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE<br />
28. Retsema, J. & Fu, W. (2001). Macrolides: structures <strong>and</strong> microbial targets. International<br />
Journal <strong>of</strong> Antimicrobial Agents (Suppl). 18, S3-10.<br />
29. Schlünzen, F., Harms, J.M., Franceschi, F., Hansen, H.A., Bartels, H., Zarivach, R. &<br />
Yonath, A. (2003). Structural basis for the antibiotic activity <strong>of</strong> ketolides <strong>and</strong> azalides. Structure<br />
(Cambridge) 11, 329-38.<br />
30. Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Peeters, J., de Kruif, A., Haesebrouck, F.<br />
(2005). The diversity <strong>of</strong> Mycoplasma hyopneumoniae within <strong>and</strong> between herds using pulsedfield<br />
gel electrophoresis. Veterinary Microbiology 109, 29-36.<br />
31. Stemke, GW., Huang, Y., Laigret, F. & Bove, J.M. (1994). Cloning the ribosomal RNA<br />
operons <strong>of</strong> Mycoplasma flocculare <strong>and</strong> comparison with those <strong>of</strong> Mycoplasma hyopneumoniae.<br />
Microbiology 140, 857-860.<br />
32. Tait-Kamradt, A., Davies, T., Cronan, M., Jacobs, M.R., Appelbaum, P.C. & Sutcliffe, J.<br />
(2000). Mutations in 23S rRNA <strong>and</strong> ribosomal protein L4 account for resistance in<br />
pneumococcal strains selected in vitro by macrolide passage. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 44, 2118-2125.<br />
33. Tanner, AC., Erickson, B.Z. & Ross, R.F. (1993). Adaptation <strong>of</strong> the Sensititre broth<br />
microdilution technique to antimicrobial susceptibility testing <strong>of</strong> Mycoplasma hyopneumoniae.<br />
Veterinary Microbiology 36, 301-306.<br />
34. Tenson, T., Lovmar, M. & Ehrenberg, M. (2003). The mechanism <strong>of</strong> action <strong>of</strong> macrolides,<br />
lincosamides <strong>and</strong> streptogramin B reveals the nascent peptide exit path in the ribosome. Journal<br />
<strong>of</strong> Molecular Biology 330, 1005-1014.<br />
35. Ter Laak, EA., Pijpers, A., Noordergraaf, J.H., Schoevers, E.C. &. Verheijden, J.H. (1991).<br />
Comparison <strong>of</strong> methods for in vitro testing <strong>of</strong> susceptibility <strong>of</strong> porcine Mycoplasma species to<br />
antimicrobial agents. Antimicrobial Agents <strong>and</strong> Chemotherapy 35, 228-233.<br />
36. Thomas, A., Nicolas, C., Dizier, I., Mainil, J. & Linden, A. (2003). Antibiotic susceptibilities<br />
<strong>of</strong> recent isolates <strong>of</strong> Mycoplasma bovis in Belgium. The Veterinary Record 153, 428-431.<br />
37. Vester, B. & Douthwaite, S. (2001). Macrolide resistance conferred by base substitutions in<br />
23S rRNA. Antimicrobial Agents <strong>and</strong> Chemotherapy 45, 1-12.<br />
38. Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. & Haesebrouck, F.<br />
(2004). In vitro susceptibilities <strong>of</strong> Mycoplasma hyopneumoniae field isolates. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 48, 4470-4472.<br />
- 137 -
2.2.3 MECHANISM OF RESISTANCE AGAINST THE<br />
FLUOROQUINOLONES FLUMEQUINE AND ENROFLOXACIN IN<br />
MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES<br />
Modified from:<br />
MECHANISMS OF RESISTANCE AGAINST THE FLUOROQUINOLONES FLUMEQUINE AND<br />
ENROFLOXACIN IN MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES<br />
J. VICCA, D. MAES, T. STAKENBORG, P. BUTAYE, F.C. MINION, J. PEETERS, A. DE KRUIF & F.<br />
HAESEBROUCK<br />
IN PREPARATION<br />
139
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
SUMMARY<br />
Ten Mycoplasma hyopneumoniae field isolates that were either sensitive (5) or<br />
resistant (5) to the fluoroquinolones flumequine <strong>and</strong> enr<strong>of</strong>loxacin, two frequently used<br />
antibiotics in swine, were selected. Their quinolone resistance-determining regions<br />
(QRDR) <strong>of</strong> gyrA, gyrB, parC <strong>and</strong> parE were characterized. Parts <strong>of</strong> the DNA gyrase<br />
subunits, gyrA <strong>and</strong> gyrB, <strong>and</strong> the topoisomerase subunits, parC <strong>and</strong> parE, containing the<br />
QRDR, were sequenced. In all 5 resistant isolates one point mutation (C → A) in parC<br />
was found, resulting in an amino acid change from serine to tyrosine at position 80 (E.<br />
coli numbering). For 4 <strong>of</strong> these isolates, this was the only mutation found. These isolates<br />
had a MIC <strong>of</strong> enr<strong>of</strong>loxacin <strong>of</strong> 0.5 µg/ml, while for sensitive isolates the MIC <strong>of</strong><br />
enr<strong>of</strong>loxacin was ≤ 0.06 µg/ml. One resistant isolate (Mh 20) had an extra mutation (C →<br />
T) in gyrA resulting in an amino acid change from alanine to valine at position 83 (E. coli<br />
numbering), correlated with an increase in the MIC <strong>of</strong> enr<strong>of</strong>loxacin (> 1 µg/ml). No<br />
mutations resulting in an amino acid change were detected in the QRDR <strong>of</strong> the gyrB <strong>and</strong><br />
parE genes <strong>of</strong> the selected isolates. This is the first description <strong>of</strong> the mechanism <strong>of</strong><br />
resistance against fluoroquinolones in M. hyopneumoniae.<br />
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2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
INTRODUCTION<br />
Mycoplasma hyopneumoniae is a major swine pathogen causing enzootic<br />
pneumonia, a chronic respiratory disease in pigs resulting in considerable economic<br />
losses. In a previous study (Vicca et al., 2004) conducted to determine the in vitro<br />
susceptibility <strong>of</strong> M. hyopneumoniae field isolates to frequently used antimicrobials in<br />
swine, 5 out <strong>of</strong> 21 isolates were found to be less susceptible or resistant to flumequine<br />
<strong>and</strong> enr<strong>of</strong>loxacin. This rather high frequency was unexpected since fluoroquinolone<br />
resistance does not <strong>of</strong>ten occur in swine respiratory pathogens (Hannan et al., 1997;<br />
Martel et al., 2001; Wallmann et al., 2003).<br />
Fluoroquinolones are broad-spectrum antimicrobials. Their use depends on the<br />
country regulations; fluoroquinolones are not allowed for use in pigs in the US but are<br />
allowed in the EU (World <strong>Health</strong> Organization, 1998). In Belgian pig herds,<br />
fluoroquinolones are frequently used as a prophylactic antibiotic during the suckling<br />
period mainly to prevent neonatal diarrhea (Timmerman et al., 2005). In older pigs, these<br />
antimicrobials are mainly used to parenterally treat individual animals with diarrhea,<br />
arthritis, meningitis or respiratory symptoms. The most frequently used fluoroquinolones<br />
in large animal veterinary medicine are flumequine <strong>and</strong> enr<strong>of</strong>loxacin.<br />
Fluoroquinolones are known to have two enzyme targets in the bacterial cell<br />
belonging to the topoisomerases type 2, namely DNA gyrase <strong>and</strong> topoisomerase IV. The<br />
first enzyme catalyses ATP-dependent negative supercoiling <strong>of</strong> DNA, the latter enzyme<br />
is essential for chromosome segregation (Gellert, 1981; Hooper, 2000). DNA gyrase is a<br />
tetramer composed <strong>of</strong> 2 GyrA <strong>and</strong> 2 GyrB subunits. Topoisomerase IV is similarly<br />
structured <strong>and</strong> is composed <strong>of</strong> 2 ParC <strong>and</strong> 2 ParE subunits. ParC is homologous to GyrA<br />
<strong>and</strong> ParE is homologous to GyrB. The primary target for fluoroquinolones in Gramnegative<br />
bacteria is the DNA gyrase, whereas in Gram-positive, including mycoplasmas,<br />
it seems to be the topoisomerase IV (Gellert, 1981; Bell<strong>and</strong> et al., 1994; Khordusky et al.,<br />
1995; Georgiou et al., 1996; Deguchi et al., 1997; Bébéar et al., 1998; Hooper, 2000;<br />
Beaucheron et al., 2004). However, some exceptions to this rule were found in<br />
Streptococcus pneumoniae <strong>and</strong> Mycoplasma hominis isolates for newer fluoroquinolones,<br />
such as sparfloxacin <strong>and</strong> gatifloxacin (Fukuda <strong>and</strong> Hiramatsu, 1999; Bébéar et al., 2000).<br />
For Mycoplasma gallisepticum, the preferential target <strong>of</strong> enr<strong>of</strong>loxacin is DNA gyrase<br />
(Reinhardt et al., 2002b). In several bacteria, mutations responsible for an increase in<br />
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2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
MIC-value were found in the subunits <strong>of</strong> these target genes (Yoshida et al., 1990;<br />
Yoshida et al., 1993; Bébéar et al., 1998; Bébéar et al., 1999; Reinhardt et al., 2002a).<br />
Resistance to fluoroquinolones has recently been described in M. hyopneumoniae<br />
(Vicca et al., 2004). The resistance mechanism, however, was unknown. In this study, the<br />
quinolone resistance determining regions (QRDR) <strong>of</strong> gyrA, gyrB, parC <strong>and</strong> parE were<br />
examined for the existence <strong>of</strong> point mutations related to resistance to fluoroquinolones.<br />
MATERIALS AND METHODS<br />
M. hyopneumoniae isolates<br />
The ten M. hyopneumoniae field isolates selected for this study were obtained<br />
between 2000 <strong>and</strong> 2002 from slaughter pigs from 10 different Belgian farrow-to-finish<br />
pig herds <strong>and</strong> were previously used for MIC determination (Vicca et al., 2004). Isolate<br />
selection for this study was based on the MIC value: 5 isolates with the highest <strong>and</strong> 5<br />
isolates with the lowest MIC values for flumequine <strong>and</strong> enr<strong>of</strong>loxacin were retained. The<br />
MIC values <strong>of</strong> flumequine <strong>and</strong> enr<strong>of</strong>loxacin for 4 isolates (Mh 4, 8, 14 <strong>and</strong> 17) were > 16<br />
µg/ml <strong>and</strong> 0.5 µg/ml, respectively. For isolate Mh 20, the MIC <strong>of</strong> flumequine was > 16<br />
µg/ml <strong>and</strong> that <strong>of</strong> enr<strong>of</strong>loxacin > 1 µg/ml. The other five isolates (Mh 7, 10, 11, 15 <strong>and</strong><br />
19) were susceptible to flumequine (MIC ≤ 2 µg/ml) <strong>and</strong> enr<strong>of</strong>loxacin (MIC ≤ 0.06<br />
µg/ml).<br />
DNA extraction <strong>and</strong> PCR amplification<br />
The M. hyopneumoniae isolates were grown in non-selective Friis medium <strong>and</strong><br />
subsequently centrifuged at 5000 g for 10 minutes. DNA was extracted using the DNeasy<br />
Tissue kit (Qiagen, Westburg, Leusden, The Netherl<strong>and</strong>s), according to the<br />
manufacturers’ instructions. Without further purification, an aliquot <strong>of</strong> the supernatant<br />
containing DNA was used as a template for PCR amplification.<br />
To sequence parts <strong>of</strong> the DNA gyrase subunits, gyrA <strong>and</strong> gyrB, <strong>and</strong> the<br />
topoisomerase subunits, parC <strong>and</strong> parE, containing the QRDR, primers were designed<br />
based on the M. hyopneumoniae genome sequence <strong>of</strong> reference strain 232 (Minion et al.,<br />
2004). Oligonucleotides MhgyrAfor (5’-CTKCCRGATGTCCGWGATGG-3’)<strong>and</strong><br />
MhgyrArev (5’-GTSGGRAARTCYGGCYCCGG-3’) were used to amplify a 557-bp<br />
143
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
gyrA fragment between positions 487 to 1043 (E. coli coordinates). A 937-bp gyrB<br />
fragment between positions 1994 to 3437 (E. coli coordinates) was amplified with<br />
primers MhgyrBfor (5’-ACATTCATAACCCTGAAGGC-3’) <strong>and</strong> MhgyrBrev (5’-<br />
GTCTCTCAAAGTTGTTCCGG-3’). To amplify the QRDR <strong>of</strong> parC, primers<br />
MhparCfor (5’-ATTCAGTAATTAATTCCCGG-3’) <strong>and</strong> MhparCrev (5’-<br />
TCTTCAAGGTAAATTTGCTG-3’) were selected to amplify a 1309-bp fragment<br />
between positions 19 to 1313 (E. coli coordinates) <strong>and</strong> a 735-bp parE fragment between<br />
positions 1046 to 1765 (E. coli coordinates) was amplified using the primers MhparEfor<br />
(5’-ATTCTTGAATTTGTTGGGC-3’) <strong>and</strong> MhparErev (5’-<br />
CCCAAGTCCTTTATAGCGC-3’). DNA amplification was performed with a DNA<br />
thermal cycler (model 9600 GeneAmp PCR system, Perkin-Elmer, Zaventem, Belgium).<br />
Each 50 µl PCR mixture contained 25 µl Mastermix (Invitrogen, Belgium), 2 µM <strong>of</strong> both<br />
primers <strong>and</strong> 2.5 µl DNA sample. Water was added to a total volume <strong>of</strong> 50 µl. For all<br />
amplification reactions, the same PCR running conditions were used, consisting <strong>of</strong> an<br />
initial cycle <strong>of</strong> 5 min denaturation at 94°C, followed by 35 cycles <strong>of</strong> 1 min denaturation<br />
at 94°C, 1 min <strong>of</strong> annealing at 55°C <strong>and</strong> 1 min <strong>of</strong> elongation at 72°C. After amplification,<br />
5 µl amplicon was mixed with 3 µl sample buffer (50% glycerol, 1mM cresol red). This<br />
mixture was electrophoresed in a 1.5% agarose gel for 75 min at 175 V in 0.5 x TBE<br />
(0.45 M Tris-HCl, 0.45 M boric acid, 0.01 M EDTA).<br />
Sequencing<br />
After purification <strong>of</strong> the PCR product with the Qiaquick PCR purification kit<br />
(Qiagen, Westburg, Leusden, The Netherl<strong>and</strong>s), both str<strong>and</strong>s <strong>of</strong> the PCR product were<br />
sequenced using the Big Dye Terminator v3-1 cycle sequencing kit (Applied Biosystems,<br />
Lennik, Belgium) on a ABI Prism 310 Genetic Analyser. The electropherograms were<br />
exported <strong>and</strong> converted to the sequence analysis s<strong>of</strong>tware, Kodon ® (Applied Maths, Sint-<br />
Martens-Latem, Belgium). The nucleic acid sequences <strong>of</strong> the QRDR <strong>of</strong> gyrA, gyrB, parC<br />
<strong>and</strong> parE <strong>of</strong> the susceptible M. hyopneumoniae isolates were compared with those <strong>of</strong> the<br />
isolates with a MIC <strong>of</strong> > 16 µg/ml <strong>and</strong> ≥ 0.5 µg/ml for flumequine <strong>and</strong> enr<strong>of</strong>loxacin,<br />
respectively (Figure 1 <strong>and</strong> 2). The deduced amino acid sequences <strong>of</strong> susceptible isolate<br />
Mh 7 <strong>and</strong> resistant isolate Mh 20 isolate were compared with the sequences <strong>of</strong><br />
Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli <strong>and</strong> to date fully<br />
sequenced human <strong>and</strong> veterinary Mycoplasma species (Figure 3). The percentage <strong>of</strong><br />
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2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
identity between the susceptible Mh 7 isolate, the other organisms <strong>and</strong> the GenBank<br />
accession numbers are listed in Table 2.<br />
RESULTS<br />
PCR amplification <strong>and</strong> sequences <strong>of</strong> PCR products<br />
Each <strong>of</strong> the selected forward <strong>and</strong> reverse primer pairs amplified one PCR product. The<br />
deduced amino acid sequences <strong>of</strong> the partial gyrA, gyrB, parC, <strong>and</strong> parE region are<br />
presented in Figures 1 <strong>and</strong> 2. An acquired C264A transition (E. coli numbering) was<br />
found in the parC gene <strong>of</strong> all 5 isolates with MICs <strong>of</strong> > 16 µg/ml <strong>and</strong> ≥ 0.5 µg/ml for<br />
flumequine <strong>and</strong> enr<strong>of</strong>loxacin, respectively. This corresponds to an amino acid change<br />
from serine to tyrosine at position 80 (E. coli numbering). An additional transition was<br />
found in isolate Mh 20. The MIC <strong>of</strong> enr<strong>of</strong>loxacin for this isolate was > 1 µg/ml, while it<br />
was 0.5 µg/ml for the other resistant isolates. This additional transition, C635T, was<br />
found in gyrA, resulting in an amino acid change from alanine to valine at position 83 (E.<br />
coli numbering) (Table 1). In the same isolate, another substitution was found in gyrA:<br />
T630A. However, this substitution did not result in an amino acid change. Other silent<br />
substitutions in the QRDR <strong>of</strong> gyrA were found in isolate Mh 7 (G651A) <strong>and</strong> in isolates<br />
Mh 15, 19 <strong>and</strong> 20 (G759A). In the QRDR <strong>of</strong> gyrB, silent substitutions were found in<br />
isolates Mh 4, 8, 11, 14, 17 <strong>and</strong> 20 (T2529A) <strong>and</strong> isolates Mh 10 <strong>and</strong> 19 (G2577A). No<br />
silent substitutions were found in the QRDR <strong>of</strong> parC. In the QRDR <strong>of</strong> parE two silent<br />
substitutions were found: C315T in isolates Mh 7 <strong>and</strong> 15, <strong>and</strong> G345A in isolates Mh 7,<br />
11 <strong>and</strong> 15. The identity for the QRDR <strong>of</strong> the 4 fluoroquinolone target genes at DNA level<br />
was very high for all M. hyopneumoniae isolates; 96.30%, 98.80%, 98.78% <strong>and</strong> 97.53%<br />
for the QRDR <strong>of</strong> gyrA, gyrB, parC <strong>and</strong> parE, respectively.<br />
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2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
Table 1: MICs for flumequine <strong>and</strong> enr<strong>of</strong>loxacin <strong>and</strong> the amino acid mutations in gyrA<br />
<strong>and</strong> parC <strong>of</strong> the fluoroquinolone resistant field isolates <strong>of</strong> M. hyopneumoniae.<br />
MIC (µg/ml)<br />
Amino acid change (codon)<br />
Isolate Flumequine Enr<strong>of</strong>loxacin gyrA parC<br />
83 * 80<br />
Mh 7 2 0.06 - -<br />
Mh 10 2 0.06 - -<br />
Mh 11 1 0.03 - -<br />
Mh 15 2 0.06 - -<br />
Mh 19 2 0.06 - -<br />
Mh 4 > 16 0.5 - S(TCT)→Y(TAT)<br />
Mh 8 > 16 0.5 - S(TCT)→Y(TAT)<br />
Mh 14 > 16 0.5 - S(TCT)→Y(TAT)<br />
Mh 17 > 16 0.5 - S(TCT)→Y(TAT)<br />
Mh 20 > 16 > 1 A(GCT)→V(GTT) S(TCT)→Y(TAT)<br />
* : amino acid position according to E. coli numbering<br />
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2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
QRDR <strong>of</strong> gyrA<br />
Mh 4 517 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 7 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 8 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 10 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 11 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 14 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 15 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 17 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 19 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 20 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT<br />
Mh 4 607 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 7 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCAATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 8 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 10 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 11 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 14 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 15 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 17 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 19 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
Mh 20 CTTGGAAAATACCATCCTCATGGAGATGTTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT<br />
* * *<br />
Mh 4 697 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG 759<br />
Mh 7 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG<br />
Mh 8 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG<br />
Mh 10 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG<br />
Mh 11 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG<br />
Mh 14 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG<br />
Mh 15 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAA<br />
Mh 17 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG<br />
Mh 19 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAA<br />
Mh 20 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAA<br />
*<br />
QRDR <strong>of</strong> gyrB<br />
Mh 4 2431 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 7 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 8 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 10 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 11 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 14 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 15 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 17 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 19 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 20 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG<br />
Mh 4 2521 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 7 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 8 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 10 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAAATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 11 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 14 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 15 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 17 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 19 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAAATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
Mh 20 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA<br />
* *<br />
Mh 4 2611 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT 2679<br />
Mh 7 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 8 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 10 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 11 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 14 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 15 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 17 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 19 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Mh 20 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT<br />
Figure 1: DNA sequence <strong>of</strong> the quinolone resistance determining region (QRDR) <strong>of</strong> the<br />
gyrA <strong>and</strong> gyrB subunit <strong>of</strong> DNA gyrase <strong>of</strong> 10 Mycoplasma hyopneumoniae field isolates.<br />
Silent mutations are underlined, substitutional mutations are underlined <strong>and</strong> bold. Nucleic<br />
acid position according to E. coli numbering.<br />
147
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
QRDR <strong>of</strong> parC<br />
Mh 4 146<br />
Mh 7<br />
Mh 8<br />
Mh 10<br />
Mh 11<br />
Mh 14<br />
Mh 15<br />
Mh 17<br />
Mh 19<br />
Mh 20<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA<br />
Mh 4 236 ATCGGAAAATATCATCCTCATGGTGATTATTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 7 ATCGGAAAATATCATCCTCATGGTGATTCTTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 8 ATCGGAAAATATCATCCTCATGGTGATTATTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 10 ATCGGAAAATATCATCCTCATGGTGATTCTTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 11 ATCGGAAAATATCATCCTCATGGTGATTCTTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 14 ATCGGAAAATATCATCCTCATGGTGATTATTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 15 ATCGGAAAATATCATCCTCATGGTGATTCTTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 17 ATCGGAAAATATCATCCTCATGGTGATTATTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 19 ATCGGAAAATATCATCCTCATGGTGATTCTTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
Mh 20 ATCGGAAAATATCATCCTCATGGTGATTATTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG<br />
*<br />
Mh 4 326 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG 391<br />
Mh 7 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
Mh 8 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
Mh 10 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
Mh 11 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
Mh 14 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
Mh 15 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
Mh 17 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
Mh 20 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG<br />
QRDR <strong>of</strong> parE<br />
Mh 4 1298 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 7 CGCGAGCTTTTTTTGGTTGAAGGTGAATCGGCAGGTGGTTCTGCAAAACTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 8 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 10 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 11 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAACTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 14 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 15 CGCGAGCTTTTTTTGGTTGAAGGTGAATCGGCAGGTGGTTCTGCAAAACTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 17 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 19 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
Mh 20 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA<br />
* *<br />
Mh 4 1388 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 7 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 8 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 10 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 11 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 14 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 15 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 17 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 19 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 20 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA<br />
Mh 4 1478 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC 1540<br />
Mh 7 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
Mh 8 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
Mh 10 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
Mh 11 ATTGGCCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATTATGACTGACGCTGAC<br />
Mh 14 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
Mh 15 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
Mh 17 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
Mh 19 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
Mh 20 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC<br />
*<br />
Figure 2: DNA sequence <strong>of</strong> the quinolone resistance determining region (QRDR) <strong>of</strong> the<br />
parC <strong>and</strong> parE subunit <strong>of</strong> topoisomerase IV <strong>of</strong> 10 Mycoplasma hyopneumoniae field<br />
isolates. Silent mutations are underlined, substitutional mutations are underlined <strong>and</strong><br />
bold. Nucleic acid position according to E. coli numbering.<br />
148
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
DISCUSSION<br />
Resistance in M. hyopneumoniae field isolates was first described in a previous<br />
study where 5 <strong>of</strong> 21 isolates were found to be resistant to flumequine <strong>and</strong> less susceptible<br />
or resistant to enr<strong>of</strong>loxacin (Vicca et al., 2004). This rather high prevalence <strong>of</strong><br />
fluoroquinolone resistance does not agree with the resistance rate found in other bacterial<br />
swine pathogens like Streptococcus suis (Martel et al., 2001; Aarestrup et al., 1998),<br />
Arcanobacterium pyogenes (Yoshimura et al., 2000), Pasteurella multocida <strong>and</strong><br />
Mannheimia haemolytica (Wallmann et al., 2003). Mycoplasma hyosynoviae <strong>and</strong><br />
Mycoplasma hyorhinis however, two other pathogenic mycoplasmas in swine, appear to<br />
exhibit a high resistance rate against flumequine (100% <strong>and</strong> 85% resistance, respectively)<br />
but are fully susceptible to enr<strong>of</strong>loxacin (Hannan et al., 1997).<br />
Several mechanisms for fluoroquinolone resistance have been described in<br />
different bacterial species. These include alterations in the two drug target enzymes,<br />
namely DNA gyrase <strong>and</strong> topoisomerase IV, changes in drug permeation through<br />
modifications in the outer membrane proteins, induction <strong>of</strong> active efflux systems,<br />
modifications in the peptidoglycan layer or the outer membrane proteins (Nikaido <strong>and</strong><br />
Thanassi, 1993; Kaatz et al., 2002) <strong>and</strong> plasmid-correlated quinolone resistance<br />
(Martinéz-Martinéz et al., 1998; Hawkey, 2003). Although the existence <strong>of</strong> energy<br />
dependent efflux systems has recently been described for M. hominis (Raherison et al.,<br />
2002), acquired resistance in mycoplasma species is usually due to alterations in the<br />
target enzymes. In the present study, the QRDR <strong>of</strong> the four target genes gyrA, gyrB, parC<br />
<strong>and</strong> parE were sequenced in fluoroquinolone susceptible <strong>and</strong> resistant M. hyopneumoniae<br />
isolates. The amino acid change at position 80 (E. coli numbering) in ParC, observed in<br />
all 5 resistant isolates, is the most common mutation related to fluoroquinolone resistance<br />
in Gram-positive bacteria (Ferrero et al., 1995; Ng et al., 1996; Yamagishi et al., 1996;<br />
Kanematsu et al., 1998), including M. hominis (Bébéar et al., 2003). For four <strong>of</strong> the M.<br />
hyopneumoniae isolates, this was the only mutation found <strong>and</strong> it resulted in at least an 8-<br />
fold increase in MIC <strong>of</strong> flumequine <strong>and</strong> enr<strong>of</strong>loxacin. Such isolates are considered to be<br />
resistant to flumequine (MIC > 16 µg/ml) (Hannan et al., 1997), while they are still<br />
considered to be susceptible to enr<strong>of</strong>loxacin (MIC = 0.5 µg/ml) (NCCLS, 2002).<br />
One isolate Mh 20 had an extra mutation (C → T) in gyrA at position 635,<br />
resulting in an amino acid change from alanine to valine at position 83 (E. coli<br />
149
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
numbering). This is associated with at least a 4-fold increase in MIC <strong>of</strong> enr<strong>of</strong>loxacin<br />
(MIC > 1 µg/ml) compared to isolates with only a mutation in parC. This location is<br />
another hot spot for fluoroquinolone resistance (Hooper, 1999).<br />
The occurrence <strong>of</strong> low-level resistance against fluoroquinolones after a single<br />
mutation in parC has earlier been described for Enterococcus faecalis, Staphylococcus<br />
aureus <strong>and</strong> Streptococcus pneumoniae, whereas high-level resistant isolates had<br />
mutations in both parC <strong>and</strong> gyrA (Kanematsu et al., 1998; Schmitz et al., 1998; Davies<br />
<strong>and</strong> Goldschmidt, 2002). In M. bovirhinis, however, a single mutation in parC (position<br />
80) resulted in different MIC pr<strong>of</strong>iles including low- <strong>and</strong> high-level resistant isolates<br />
(Hirose et al., 2004). The authors suggested that the differences in MIC might have been<br />
caused by the level <strong>of</strong> expression <strong>of</strong> the quinolone efflux transporter.<br />
As in fluoroquinolone resistant M. hominis, Ureaplasma urealyticum <strong>and</strong><br />
Acholeplasma laidlawii isolates, no mutations were found in the QRDR <strong>of</strong> gyrB in M.<br />
hyopneumoniae. Such mutations have been described in in vitro selected resistant M.<br />
gallisepticum isolates (Reinhardt et al., 2002a). Also, no mutations resulting in amino<br />
acid changes were found in the QRDR <strong>of</strong> parE <strong>of</strong> the M. hyopneumoniae isolates. In<br />
clinical isolates <strong>of</strong> M. hominis however, a mutation resulting in an amino acid<br />
substitution in parE was previously observed (Bébéar et al., 1999). The absence <strong>of</strong> amino<br />
acid changes in GyrB <strong>and</strong> ParE <strong>of</strong> fluoroquinolone resistant M. hyopneumoniae isolates is<br />
in agreement with other studies reporting that amino acid changes in GyrB or ParE occur<br />
less frequently than in GyrA <strong>and</strong> ParC (Hooper, 1999).<br />
CONCLUSION<br />
Topoisomerase IV <strong>of</strong> M. hyopneumoniae seems the primary target for fluoroquinolones<br />
(flumequine <strong>and</strong> enr<strong>of</strong>loxacin) with position 80 in parC as the hot spot. A single mutation<br />
in parC is sufficient to reach resistance to flumequine, while a second mutation in the<br />
secondary target, DNA gyrase (gyrA), is necessary to make M. hyopneumoniae resistant<br />
to enr<strong>of</strong>loxacin.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by the Federal Public Service <strong>of</strong> Public <strong>Health</strong>, Food<br />
Chain Security <strong>and</strong> Environment, Brussels, Belgium, grant no. S-6039.<br />
150
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
151
GyrA<br />
Mh 7<br />
VHRRILYTMAELGITSGTSYKKSARIVGDVLGKYHPHGDASVYESMVRMAQPFSLRYPLVDGHGNFGSIDGDEAAAMRYTE<br />
Mh 20 VHRRILYTMAELGITSGTSYKKSARIVGDVLGKYHPHGDVSVYESMVRMAQPFSLRYPLVDGHGNFGSIDGDEAAAMRYTE<br />
Mhom<br />
VHRRILYGMSELGMFYTAPHKKSARIVGDVLGKYHPHGDSSVYEAMVRMAQDFSLRYPLIDGHGNFGSVDGDEAAAMRYTE<br />
Mgen<br />
VHRRVLYGAYIGGMHHDRPFKKSARIVGDVMSKFHPHGDMAIYDTMSRMAQDFSLRYLLIDGHGNFGSIDGDRPAAQRYTE<br />
Mgal<br />
PVTWVLYGAYTSGLTHDKPYRKSAQIVGHVMGKYHPHGDSAIYETMVRMAQPFSLRYMLIDGHGNFGSIDGDSAGAMRYTE<br />
Mmob<br />
VHRRILYGMTELGLFYNAQYKKSARVVGDVLGKYHPHGDSSVYEAMVRMAQDFSLRYPLIDGHGNFGSIDGDSAAAMRYTE<br />
Mmyc<br />
VHRRIIYAMNDLGITSDKPHKKSARIVGEVIGKYHPHGDSAVYETMVRMAQEFSYRYPLIDGHGNFGSIDGDGAAAMRYTE<br />
Mpen<br />
VHRRVLYAAYNMGMTHDKPYKKSARLVGEVIGKYHPHGDTAAYETMVRMAQDFSMRYMLVDGHGNFGSIDGDSAAAMRYTE<br />
Mpneu VHRRVLYGAYTGGMHHDRPFKKSARIVGDVMSKFHPHGDMAIYDTMSRMAQDFSLRYLLIDGHGNFGSIDGDRPAAQRYTE<br />
Mpul<br />
VHRRILYGMFDLGLHHSSAFKKSARIVGDVLGKYHPHGDASVYEAMVRMAQEFSMRYPLVDGHGNFGSIDGDPAAAMRYTE<br />
Saur<br />
VHRRILYGLNEQGMTPDKSYKKSARIVGDVMGKYHPHGDSSIYEAMVRMAQDFSYRYPLVDGQGNFGSMDGDGAAAMRYTE<br />
Spneu VHRRILYGMNELGVTPDKPHKKSARITGFVMGKYHPHGDSSIYEAMVRMAQWWSYRYMLVDGHGNFGSMDGDSAAAQRYTE<br />
Ecoli 44 VHRRVLYAMNVLGNDWNKAYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTE<br />
124<br />
** * *** * * * ***** * **** * ** * ** ***** *** * ****<br />
GyrB<br />
Mh 7<br />
AELYIVEGNSAGGSAKMGRDRHFQAILPLRGKVINSQRFQLEKVLKNEEILSMITAFGTGVGP-EFDISKIRYQKIIIMTDAD<br />
Mh 20 AELYIVEGNSAGGSAKMGRDRHFQAILPLRGKVINSQRFQLEKVLKNEEILSMITAFGTGVGP-EFDISKIRYQKIIIMTDAD<br />
Mhom<br />
RELFIVEGNSAGGSAKMGRDRSIQAILPLRGKVINAEKNSFASVLSNKEIATMIHALGTGINT-EFDINKLKYHKIIIMTDAD<br />
Mgen<br />
SELYIVEGDSAGGTAKTGRDRYFQAILPLRGKILNVEKSNFEQIFNNAEISALVMAIGCGIKP-DFELEKLRYSKIVIMTDAD<br />
Mgal<br />
SELYIVEGDSAGGSAKSGRDRFYQAILPLRGKVLNVEKANHEKIFKNEEIRTLITAIGAGVNP-EFSLDKIRYNKIIIMTDAD<br />
Mmob<br />
SELYIVEGNSAGGSAKMGRDRETQAILPLRGKVINAEKANIVKVFENTEILSLITALGTGVGP-EFNINKLRYHKVVIMTDAD<br />
Mmyc<br />
AELYLVEGDSAGGSAKTGRNRKFQAILPLRGKVLNVERVTEARAFSNNEIKSIITAIGTGIKE-ELDLSKLRYKKIVIMTDAD<br />
Mpen<br />
SELYIVEGDSAGGSAKLGRDRIFQAILPLKGKIINVEKAKSDKIFSNEEIINLITAIGAGVGP-EFKIDKLRYNKIILMTDAD<br />
Mpneu SELYIVEGDSAGGTAKTGRDRYFQAILPLRGKILNVEKSHFEQIFNNVEISALVMAVGCGIKP-DFELEKLRYNKIIIMTDAD<br />
Mpul<br />
SELYIVEGNSAGGSAKMGRDREFQAILPLRGKVINAEKNNIEKVFNNEEIQSLIIALGTGIAE-EFNINKLRYHKIIIMTDAD<br />
Saur<br />
CEIFLVEGDSAGGSTKSGRDSRTQAILPLRGKILNVEKARLDRILNNNEIRQMITAFGTGIGG-DFDLAKARYHKIVIMTDAD<br />
Spneu TELFIVEGDSAGGSAKSGRNREFQAILPIRGKILNVEKASMDKILANEEIRSLFTAMGTGFGA-EFDVSKARYQKLVLMTDAD<br />
Ecoli 418 SELYLVEGDSAGGSAKQGRNRKNQAILPLKGKILNVEKARFDKMLSSQEVATLITALGCGIGRDEYNPDKLRYHSIIIMTDAD 500<br />
* *** **** * ** ****** ** * * * * ** *****<br />
ParC<br />
Mh 7<br />
VQRRILYSMWQLGLKNSKNYKKSARVVGDVIGKYHPHGDSSIYDALVRLAQEWKMNSPLVEMHGNKGSIDD-DPPAAMRYTE<br />
Mh 20 VQRRILYSMWQLGLKNSKNYKKSARVVGDVIGKYHPHGDYSIYDALVRLAQEWKMNSPLVEMHGNKGSIDD-DPPAAMRYTE<br />
Mhom<br />
VQRRILYSMWNLHLKNSEPFKKSARIVGDVIGRYHPHGDSSIYEALVRMAQDWKSNFPLIEMHGNKGSIDD-DPAAAMRYTE<br />
Mgen<br />
VQRRILYGMFQMGLKPTTPYKKSARAVGEIMGKYHPHGDSSIYDAIIRMSQSWKNNWTTVSIHGNNGSVDG-DNAAAMRYTE<br />
Mgal<br />
VQRRVLYGMYNLGLYYNKSYRKSAATVGEVIGKFHPHGDSSIYEALVRMTQSWKNNIPLIDMQGNNGSIDG-DNAAAMRYTE<br />
Mmob<br />
VQRRILYSMHELGLTSEKPFKKSARVVGDVIGKYHPHGDSSIYEAMVRMSQEWKMNVPLIQMHGNIGSIDD-DPAAAMRYTE<br />
Mmyc<br />
VQRRILYAMNQLNLTFDKPYKKSARVVGEVIGKYHPHGDSSIYDAMVRMSQWWKVNIPLVDMQGNNGSIDG-DSAAAMRYTE<br />
Mpen<br />
VQRRILHAMNELKIHHDKPYKKSARTVGEVIGKYHPHGDSSIYEAMVRMSQEWKNNLPLLDMQGNKGSIDG-DSPAAMRYTE<br />
Mpneu VQRRILYGMYQMGLKPTSPYKKSARAVGEIMGKYHPHGDASIYDAIVRMSQAWKNNLTTISIHGNNGSIDG-DNAAAMRYTE<br />
Mpul<br />
VQRRILYSMMDLKLWNDKPFKKSARIVGDVIGKYHPHGDSSIYEAMVRMAQDWKMNIPLIQMHGNIGSIDD-DPAAAMRYTE<br />
Saur<br />
VQRRILYAMYSSGNTHDKNFRKSAKTVGDVIGQYHPHGDSSVYEAMVRLSQDWKLRHVLIEMHGNNGSIDN-DPPAAMRYTE<br />
Spneu VQRRILYSMNKDSNTFDKSYRKSAKSVGNIMGNFHPHGDSSIYDAMVRMSQNWKNREILVEMHGNNGSMDG-DPPAAMRYTE<br />
Ecoli 41 VQRRIVYAMSELGLNASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLVDGQGNWGAPDDPKSFAAMRYTE<br />
122<br />
**** * *** ** * ***** * * * ** * *******<br />
ParE<br />
Mh 7<br />
Mh 20<br />
Mhom<br />
Mgen<br />
Mgal<br />
Mmob<br />
Mmyc<br />
Mpen<br />
Mpneu<br />
Mpul<br />
Saur<br />
Spneu<br />
Ecoli<br />
RELFLVEGESAGGSAKLARNREFQAILPLKGKIVNAQKTR-LIDLLKNEEIIAIISALGTGIGQNFNLKNLNYGKIIIMTDAD<br />
RELFLVEGESAGGSAKLARNREFQAILPLKGKIVNAQKTR-LIDLLKNEEIIAIISALGTGIGQNFNLKNLNYGKIIIMTDAD<br />
KELFLVEGDSAGGSAKLGRNRVTQAILPLRGKVINTDKAK-LSDVLANEEIATIINTIGAGIGEDFNLKNAQYHKIIIMTDAD<br />
KELFIVEGDSAGGTAKMGRDRIFQAILPLRGKVLNVEKINNKKEAITNEEILTLIFCIGTGILTNFNIKDLKYGKIIIMTDAD<br />
NEIFLVEGDSAGGTAKSGRDKRFQAILPLRGKVVNVEKSR-LQDLLKNEEILSIISCLGCGIGNNFNIKNLKYHKIIIMTDAD<br />
KELFLVEGDSAGGSAKLGRNSQFQAILPLKGKVINCEKTK-LIDVLKNEEISTIINTIGAGIGADFDLKKANYHKVVIMTDAD<br />
NELYLVEGDSAGGSAKTGRNRKFQAILPLRGKVINSEKAK-LVDLLKNEEIQSIINAIGAGVGKDFDISDINYGKIIIMTDAD<br />
TELFLVEGDSAGGSAKLGRDKRYQAILPLKGKVINVEKAM-LKDLLKNEEISTIISSIGGSIGSDFCLPDVKYSKIIIMTDAD<br />
RELFVVEGDSAGGTAKMGRDRFLQAILPLRGKVLNVEKINNKKEAINNEELLTLIFCIGTGIGNNFTIRDRKYDKIIIMTDAD<br />
NEIFLVEGDSAGGSAKSGRNRRFQAILPLRGKVINTEKSR-LIDILKNEEISTIINALNTGVGEDFEIKNLKYHKIIIMTDAD<br />
NELYLVEGDSAGGSAKLGRDRKFQAILPLRGKVINTEKAR-LEDIFKNEEINTIIHTIGAGVGTDFKIEDSNYNRVIIMTDAD<br />
NELYLVEGDSAGGSAKQGRDRKFQAILPLRGKVINTAKAK-MADILKNEEINTMIYTIGAGVGADFSIEDANYDKIIIMTDAD<br />
412 TELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVS-SDEVLASQEVHDISVAIGIDPDSD-DLSQLRYGKICILADAD<br />
492<br />
* *** **** ** * *** ** ** * * * * ***<br />
Figure 3: Alignment <strong>of</strong> Mycoplasma hyopneumoniae GyrA, GyrB, ParC <strong>and</strong> ParE<br />
QRDR amino acid sequences <strong>of</strong> a susceptible isolate (Mh 7) with its counterpart from<br />
M. hyopneumoniae (resistant isolate, Mh 20), M. hominis (Mhom), M. genitalium<br />
(Mgen), M. gallisepticum (Mgal), M. mobile (Mmob), M. mycoides subsp. mycoides<br />
SC (Mmyc), M. penetrans (Mpen), M. pneumoniae (Mpneu), M. pulmonis (Mpul),<br />
Staphylococcus aureus (Saur), Streptococcus pneumoniae (Spneu) <strong>and</strong> Escherichia<br />
- 152 -
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
coli (Ecoli). GenBank accession numbers <strong>and</strong> percentage homology are listed in Table<br />
2. An asterisk indicates a residue identical in all 13 amino acid sequences. Dashes<br />
indicate gaps.<br />
- 153 -
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
REFERENCES<br />
1. Aarestrup, F.M., Jorsal, S.E. & Jensen, N.E. (1998). Serological characterization <strong>and</strong><br />
antimicrobial susceptibility <strong>of</strong> Streptococcus suis isolates from diagnostic samples in Denmark<br />
during 1995 <strong>and</strong> 1996. Veterinary Microbiology 60, 59-66.<br />
2. Baucheron, S., Chaslus-Dancla, E. & Cloekaert, A. (2004). Role <strong>of</strong> TolC <strong>and</strong> parC mutation<br />
in high-level fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT 204.<br />
Journal <strong>of</strong> Antimicrobial Chemotherapy 53, 657-659.<br />
3. Bébéar, C.M., Grau, O., Charron, A., Renaudin, H., Gruson, D. & Bébéar, C. (2000).<br />
Cloning <strong>and</strong> nucleotide sequence <strong>of</strong> the DNA Gyrase (gyrA) gene from Mycoplasma hominis<br />
<strong>and</strong> characterization <strong>of</strong> quinolone-resistant mutants selected in vitro with trov<strong>of</strong>loxacin.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 44, 2719-2727.<br />
4. Bébéar, C.M., Renaudin, H., Charron, A., Bové, J.M., Bébéar, C. & Renaudin, J. (1998).<br />
Alterations in topoisomerase IV <strong>and</strong> DNA gyrase in quinolone-resistant mutants <strong>of</strong> Mycoplasma<br />
hominis obtained in vitro. Antimicrobial Agents <strong>and</strong> Chemotherapy 42, 2304-2311.<br />
5. Bébéar, C.M., Renaudin, J., Charron, A, Renaudin, H., De Barbeyrac, B., Schaeverbeke,<br />
T. & Bébéar, C. (1999). Mutations in the gyrA, parC <strong>and</strong> parE genes associated with<br />
fluoroquinolone resistance in clinical isolates <strong>of</strong> Mycoplasma hominis. Antimicrobial Agents<br />
<strong>and</strong> Chemotherapy 43, 954-956.<br />
6. Bébéar, C.M., Renaudin, H., Charron, A., Clerc, M., Pereyre, S. & Bébéar, C. (2003). DNA<br />
gyrase <strong>and</strong> topoisomerase IV mutations in clinical isolates <strong>of</strong> Ureaplasma spp. <strong>and</strong> Mycoplasma<br />
hominis resistant to fluoroquinolones. Antimicrobial Agents <strong>and</strong> Chemotherapy 47, 3323-3325.<br />
7. Bell<strong>and</strong>, R.J., Morrison, S.G., Ison, C. & Huang, W.M. (1994). Neisseria gonorrhoeae<br />
acquires mutations in analogous regions <strong>of</strong> gyrA <strong>and</strong> parC in fluoroquinolone-resistant isolates.<br />
Molecular Microbiology 14, 371-380.<br />
8. Davies, T.A. & Goldschmidt, R. (2002). Screening <strong>of</strong> large numbers <strong>of</strong> Streptococcus<br />
pneumoniae isolates for mutations associated with fluoroquinolone resistance using an<br />
oligonucleotide probe assay. FEMS Microbiology Letters 217, 219-224.<br />
9. Deguchi, T., Fukuoka, A., Yasuda, M., Nakano, M., Ozeki, S., Kanematsu, E., Nishino, Y.,<br />
Ishihara, S., Ban, Y. & Kawada, Y. (1997). Alterations in the GyrA subunit <strong>of</strong> DNA gyrase<br />
<strong>and</strong> the ParC subunit <strong>of</strong> topoisomerase IV in quinolone-resistant clinical isolates <strong>of</strong> Klebsiella<br />
pneumoniae. Antimicrobial Agents <strong>and</strong> Chemotherapy 41, 699-701.<br />
10. Ferrero, L., Cameron, B. & Crouzet, J. (1995). Analysis <strong>of</strong> gyrA <strong>and</strong> grlA mutations in<br />
stepwise-selected cipr<strong>of</strong>loxacin-resistant mutants <strong>of</strong> Staphylococcus aureus. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 39, 1554-1558.<br />
11. Fukuda, H. & Hiramatsu, K. (1999). Primary targets <strong>of</strong> fluoroquinolones in Streptococcus<br />
pneumoniae. Antimicrobial Agents <strong>and</strong> Chemotherapy 43, 410-412.<br />
12. Gellert, M. (1981). DNA topoisomerases. Annual Re<strong>view</strong> <strong>of</strong> Biochemistry 50, 879-910.<br />
13. Georgiou, M., Muñoz, R., Román, F., Cantón, R., Gómez-Lus, R., Campos, J. & De La<br />
Campa, A.G. (1996). Cipr<strong>of</strong>loxacin-resistant Haemophilus influenzae strains possess mutations<br />
in analogous positions <strong>of</strong> GyrA <strong>and</strong> ParC. Antimicrobial Agents <strong>and</strong> Chemotherapy 40, 1741-<br />
1744.<br />
14. Hannan, P.C.T., Windsor, H.M., de Jong, A., Schmeer, N. & Stegemann, M. (1997).<br />
Comparitive susceptibilities <strong>of</strong> various animal-pathogenic mycoplasmas to fluoroquinolones.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 41, 2037-2040.<br />
15. Hawkey, P.M. (2003). Mechanism <strong>of</strong> quinolone action <strong>and</strong> microbial response. Journal <strong>of</strong><br />
Antimicrobial Chemotherapy (Suppl. 1) 51, 29-35.<br />
- 154 -
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
16. Hirose, K., Kawasaki, Y. & Kotani, K. (2004). Characterization <strong>of</strong> a point mutation in the<br />
parC gene <strong>of</strong> Mycoplasma bovirhinis associated with fluoroquinolone resistance. Journal <strong>of</strong><br />
Veterinary Medicine, Series B 51, 169-75.<br />
17. Hooper, D.C. (1999). Mechanisms <strong>of</strong> fluoroquinolone resistance. Drug Resistance Updates 2,<br />
38-55.<br />
18. Hooper, D.C. (2000). Mechanism <strong>of</strong> action <strong>and</strong> resistance <strong>of</strong> older <strong>and</strong> newer fluoroquinolones.<br />
Clinical Infectious Diseases (Suppl. 2) 31, 24-28.<br />
19. Kaatz, G.W., Moudgal, V.V. & Seo, S.M. (2002). Identification <strong>and</strong> characterization <strong>of</strong> a novel<br />
efflux-related multidrug resistance phenotype in Staphylococcus aureus. Journal <strong>of</strong><br />
Antimicrobial Chemotherapy 50, 833-838.<br />
20. Kanematsu, E., Deguchi, T., Yasuda, M., Kawamura, T., Nishino, Y. & Kawada, Y. (1998).<br />
Alterations in the GyrA subunit <strong>of</strong> DNA gyrase <strong>and</strong> the ParC subunit <strong>of</strong> DNA topoisomerase IV<br />
associated with quinolone resistance in Enterococcus faecalis. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 42, 433-435.<br />
21. Khodursky, A.B., Zechiedrich, E.L. & Cozzarelli, N.R. (1995). Topoisomerase IV is a target<br />
<strong>of</strong> quinolones in Escherichia coli. Proceedings <strong>of</strong> the National Academy <strong>of</strong> Science <strong>of</strong> the<br />
United States <strong>of</strong> America 92, 11801-11802.<br />
22. Martel, A., Baele, M., Devriese, L. A., Goossens, H., Wisselink, H.J., Decostere, A. &<br />
Haesebrouck, F. (2001). Prevalence <strong>and</strong> mechanism <strong>of</strong> resistance against macrolides <strong>and</strong><br />
lincosamides in Streptococcus suis isolates. Veterinary Microbiology 83, 287-297.<br />
23. Martinéz-Martinéz, L., Pascual, A. & Jacoby, G. A. (1998). Quinolone resistance from a<br />
transferable plasmid. The Lancet 335, 797-99.<br />
24. Minion, F.C., Lefkowitz, E.J., Madsen, M.L., Cleary, B.J., Swartzell, S.M. & Mahairas,<br />
G.G. (2004). The genome sequence <strong>of</strong> Mycoplasma hyopneumoniae strain 232, the agent <strong>of</strong><br />
swine mycoplasmosis. Journal <strong>of</strong> Bacteriology 186, 7123-7133.<br />
25. National Committee for Clinical Laboratory St<strong>and</strong>ards (NCCLS). (2002). Performance<br />
st<strong>and</strong>ards for antimicrobial disk <strong>and</strong> dilution susceptibility tests for bacteria isolated from<br />
animals; approved st<strong>and</strong>ard, 2nd edn. NCCLS document M31-A2, Wayne, PA 19087, U.S.A.<br />
26. Ng, E.Y., Trucksis, M. & Hooper, D.C. (1996). Quinolone resistance mutations in<br />
topoisomerase IV: relationship <strong>of</strong> the flqA locus <strong>and</strong> genetic evidence that topoisomerase IV is<br />
the primary target <strong>of</strong> fluoroquinolones in Staphylococcus aureus. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 40, 1881-1888.<br />
27. Nikaido, H. & Thanassi, D.G. (1993). Penetration <strong>of</strong> lipophilic agents with multiple<br />
protonation sites into bacterial cells: tetracyclines <strong>and</strong> fluoroquinolones as examples.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 37, 1393-1399.<br />
28. Raherison, S., Gonzales, P., Renaudin, H., Charron, A., Bébéar, C. & Bébéar, C.M. (2002).<br />
Evidence <strong>of</strong> active efflux in resistance to cipr<strong>of</strong>loxacin <strong>and</strong> to ethidium bromide by Mycoplasma<br />
hominis. Antimicrobial Agents <strong>and</strong> Chemotherapy 46, 672-679.<br />
29. Reinhardt, A.K., Bébéar, C.M., Kobisch, M., Kempf, I. & Gautier-Bouchardon, A.V.<br />
(2002a). Characterization <strong>of</strong> mutations in DNA gyrase <strong>and</strong> topoisomerase IV involved in<br />
quinolone resistance <strong>of</strong> Mycoplasma gallisepticum mutants obtained in vitro. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 46, 590-593.<br />
30. Reinhardt, A.K., Kempf, I., Kobisch, M. & Gautier-Bouchardon, A.V. (2002b).<br />
Fluoroquinolone resistance in Mycoplasma gallisepticum: DNA gyrase as primary target <strong>of</strong><br />
enr<strong>of</strong>loxacin <strong>and</strong> impact <strong>of</strong> mutations in topoisomerases on resistance level. Journal <strong>of</strong><br />
Antimicrobial Chemotherapy 50, 589-592.<br />
31. Schmitz, F.J., Jones, M.E., H<strong>of</strong>mann, B., Scheuring, S., Lückefahr, M., Fluit, A., Verhoef,<br />
J., Hadding, U., Heinz, H-P. & Köhrer, K. (1998). Characterization <strong>of</strong> grlA, grlB, gyrA, <strong>and</strong><br />
- 155 -
2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES<br />
gyrB mutations in 116 unrelated isolates <strong>of</strong> Staphylococcus aureus <strong>and</strong> effects <strong>of</strong> mutations on<br />
cipr<strong>of</strong>loxacin MIC. Antimicrobial Agents <strong>and</strong> Chemotherapy 42, 1249-1252.<br />
32. Yamagishi, J-I., Kojima, T., Oyamada, Y., Fujimoto, K., Hattori, H., Nakamura, S. &<br />
Inoue M. (1996). Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone<br />
resistance in Staphylococcus aureus. Antimicrobial Agents <strong>and</strong> Chemotherapy 40, 1157-1163.<br />
33. Timmerman, T., Dewulf, J., Catry, B., Feyen, B., Opsomer, G., de Kruif, A. & Maes, D.<br />
(2005). Quantification <strong>and</strong> evaluation <strong>of</strong> antimicrobial-drug use in group treatments for fattening<br />
pigs in Belgium. Preventive Veterinary Medicine, accepted.<br />
34. Vicca, J., Stakenborg T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. & Haesebrouck, F.<br />
(2004). In vitro susceptibility <strong>of</strong> Mycoplasma hyopneumoniae field isolates. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 48, 4470-4472.<br />
35. Wallmann, J., Schröter, K., Wieler, L.H. & Kroker, R. (2003). National antibiotic resistance<br />
monitoring in veterinary pathogens from sick food-producing animals: the German programme<br />
<strong>and</strong> results from the 2001 pilot study. International Journal <strong>of</strong> Antimicrobial Agents 22, 420-<br />
428.<br />
36. World <strong>Health</strong> Organization. (1998). Use <strong>of</strong> Quinolones in Food Animals <strong>and</strong> Potential Impact<br />
on Human <strong>Health</strong>. [Online.] http://www.who.int/emcdocuments/zoonoses/docs/whoemczdi9810.html#Quinolone%20use%20in%20animals<br />
(15<br />
february 2005, date last accessed).<br />
37. Yoshida, H., Bogaki, M., Nakamura, M. & Nakamura, S. (1990). Quinolone resistancedeterming<br />
region in the DNA gyrase gyrA gene <strong>of</strong> Escherichia coli. Antimicrobial Agents <strong>and</strong><br />
Chemotherapy 34, 1271-1272.<br />
38. Yoshida, H., Nakamura, M., Bogaki, M., Ito, H., Kojima, T., Hattori, H. & Nakamura, S.<br />
(1993). Mechanism <strong>of</strong> action <strong>of</strong> quinolones against Escherichia coli DNA gyrase. Antimicrobial<br />
Agents <strong>and</strong> Chemotherapy 37, 839-845.<br />
39. Yoshimura, H., Kojima, A. & Ishimaru, M. (2000). Antimicrobial susceptibility <strong>of</strong><br />
Arcanobacterium pyogenes isolated from cattle <strong>and</strong> pigs. Journal <strong>of</strong> Veterinary Medicine Series<br />
B 47, 139-143.<br />
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2.2.4 THE EFFICACY OF TYLOSIN PREMIX FOR THE<br />
TREATMENT AND CONTROL OF MYCOPLASMA<br />
HYOPNEUMONIAE INFECTIONS<br />
Modified from:<br />
EFFICACY OF IN-FEED MEDICATION WITH TYLOSIN FOR THE TREATMENT AND CONTROL OF<br />
MYCOPLASMA HYOPNEUMONIAE INFECTIONS<br />
J. VICCA, D. MAES, L. JONKER, A. DE KRUIF & F. HAESEBROUCK<br />
THE VETERINARY RECORD 2005, 156, 606-610<br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
SUMMARY<br />
The efficacy <strong>of</strong> in-feed medication with tylosin (Tylan ® Premix; Elanco<br />
Animal <strong>Health</strong>) for the treatment <strong>of</strong> enzootic pneumonia was examined in an<br />
experimental Mycoplasma hyopneumoniae infection model. Three groups <strong>of</strong> 10<br />
conventional M. hyopneumoniae-free piglets were inoculated intratracheally with a<br />
highly virulent M. hyopneumoniae field isolate (non-treated control (non-TP) group<br />
<strong>and</strong> tylosin premix (TP) group) or with sterile culture medium (non-infected <strong>and</strong> nontreated<br />
control (NC) group). Twelve days after infection, tylosin treatment <strong>of</strong> the TP<br />
group was initiated (100 mg/kg <strong>of</strong> feed for 21 days). Animals were daily examined for<br />
the presence <strong>of</strong> clinical signs <strong>and</strong> a respiratory disease score (RDS) was given per pig.<br />
Thirty-three days after infection, all pigs were euthanased <strong>and</strong> the lung lesions were<br />
quantified. Lung samples were processed for immun<strong>of</strong>luorescence testing for M.<br />
hyopneumoniae. Average RDS <strong>and</strong> lung lesion scores were significantly higher<br />
(P
2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
INTRODUCTION<br />
Enzootic pneumonia, with Mycoplasma hyopneumoniae as the primary agent,<br />
remains an important health disorder in modern pig herds. Enzootic pneumonia has<br />
been demonstrated to affect animal welfare <strong>and</strong> to cause substantial economic losses,<br />
either directly or by potentiating <strong>and</strong> increasing the susceptibility <strong>of</strong> the lungs to<br />
secondary pathogens like Pasteurella multocida <strong>and</strong> Actinobacillus<br />
pleuropneumoniae (Maes et al., 1999a, 2000). M. hyopneumoniae also plays a key<br />
role in porcine respiratory disease complex, together with porcine reproductive <strong>and</strong><br />
respiratory syndrome virus, swine influenza virus <strong>and</strong> other pathogens.<br />
Control <strong>of</strong> enzootic pneumonia is largely based on good management<br />
practices, optimal housing conditions, vaccination <strong>and</strong> the use <strong>of</strong> antibiotics.<br />
Commercial M. hyopneumoniae vaccines are widely used <strong>and</strong> several studies have<br />
shown that vaccination has beneficial effects on daily weight gain (DWG) <strong>and</strong><br />
mycoplasmal pneumonia lesions (Dohoo <strong>and</strong> Montgomery, 1996; Maes et al., 1998,<br />
1999b). Compared to vaccination, the use <strong>of</strong> antibiotics in M. hyopneumoniae<br />
outbreaks has the advantage that they can be applied in a more flexible way <strong>and</strong> that<br />
they can also decrease infections with other (respiratory) bacterial pathogens.<br />
Across the European Union, tylosin is used for the treatment <strong>and</strong> control <strong>of</strong><br />
enzootic pneumonia in pigs through administration via the feed. Tylosin is a<br />
macrolide drug developed in 1961 <strong>and</strong> is exclusively used in veterinary medicine.<br />
Macrolides inhibit bacterial protein synthesis by reversibly binding to the 23 S<br />
ribosomal RNA in the 50 S subunit <strong>of</strong> the ribosome <strong>and</strong> bind at the donor site, thus<br />
preventing the translocation necessary to keep the peptide chain growing (Brisson-<br />
Noël et al., 1988). Tylosin, like other macrolides, is widely distributed throughout<br />
tissues <strong>and</strong> body fluids (Prats et al., 2002).<br />
Studies have demonstrated good efficacy <strong>of</strong> tylosin against M. hyopneumoniae<br />
in vitro (Ter Laak et al., 1991; Tanner et al., 1993; Wu et al., 1997) <strong>and</strong> in vivo<br />
(Hannan et al., 1982; Ueda et al., 1994). Previous in vivo studies were conducted with<br />
neonatal piglets inoculated with lung homogenate containing different Mycoplasma<br />
species or the preventive use <strong>of</strong> tylosin was examined. The current study was<br />
conducted in order to further examine the efficacy <strong>of</strong> tylosin when applied at the onset<br />
<strong>of</strong> clinical symptoms following infection with M. hyopneumoniae. Therefore, pigs<br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
were experimentally infected with a high dosis (7 ml <strong>of</strong> 10 7 CCU/ml) <strong>of</strong> a highly<br />
virulent M. hyopneumoniae field isolate. The currently licensed dosage <strong>of</strong> 100 mg <strong>of</strong><br />
tylosin per kg feed for 21 days was applied.<br />
MATERIALS AND METHODS<br />
M. hyopneumoniae isolate<br />
For the inoculation <strong>of</strong> the piglets, a Belgian M. hyopneumoniae field isolate<br />
was used. The isolate was obtained in 2000 from a pig herd experiencing clinical<br />
symptoms associated with enzootic pneumonia <strong>and</strong> could be filter cloned after 10<br />
passages in vitro. The isolate proved to be highly virulent during earlier comparative<br />
virulence studies (Vicca et al., 2003). The minimal inhibitory concentration <strong>of</strong> tylosin<br />
for this isolate was
2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
piglets was housed in rooms equipped with absolute filters (HEPA U15) to avoid<br />
spread <strong>of</strong> M. hyopneumoniae-organisms between groups.<br />
Immediately after weighing <strong>and</strong> allocation to the treatment groups, all pigs<br />
were anaesthetized intramuscularly with 0.22 ml/kg <strong>of</strong> a mixture <strong>of</strong> xylazin (Xyl-M ®<br />
2%; VMD) <strong>and</strong> Zolazepam <strong>and</strong> Tiletamin (Zoletil ® 100; Virbac), ear tagged <strong>and</strong><br />
inoculated intratracheally with 7 x 10 7 CCU <strong>of</strong> the M. hyopneumoniae isolate in 7 ml<br />
inoculum (non-treated control (non-TP) group <strong>and</strong> tylosin premix (TP) group) or with<br />
7 ml sterile culture medium (non-infected <strong>and</strong> non-treated control (NC) group). The<br />
inoculation day was designated as day 0 after infection.<br />
The study was conducted after approval by the Ethical Committee for animal<br />
experiments <strong>of</strong> the Faculty <strong>of</strong> Veterinary Medicine, Ghent University.<br />
Tylosin treatment<br />
None <strong>of</strong> the groups received any antimicrobials during the premedication<br />
period (0-12 days after infection). Tylosin treatment started at 12 days after infection,<br />
the day after onset <strong>of</strong> the disease (11 days after infection) when 10% <strong>of</strong> the animals<br />
over the two M. hyopneumoniae inoculated groups showed coughing. The duration <strong>of</strong><br />
the medication period was 21 days (13-33 days after infection).<br />
Blank unmedicated feed was given to the non-TP <strong>and</strong> NC group. For the TP<br />
group, a commercial batch <strong>of</strong> Tylan ® 100 Premix (tylosin 10%, 100 g/kg premix;<br />
Elanco Animal <strong>Health</strong>) was used. One gram Tylan ® Premix was mixed per kg blank<br />
unmedicated feed, this was equivalent to approximately 3-6 mg tylosin/kg<br />
bodyweight. At the beginning <strong>of</strong> the study, the presence <strong>of</strong> the appropriate tylosin<br />
level in a pooled sample was confirmed to be 99.6 mg/kg feed.<br />
Clinical evaluation <strong>and</strong> performance parameters<br />
For a period <strong>of</strong> 33 days following challenge, pigs were observed daily for 25<br />
minutes, to evaluate demeanour, abdominal filling (gauntness), presence <strong>of</strong> dyspnoea<br />
<strong>and</strong> tachypnoea <strong>and</strong> behavioural changes. In addition, a clinical respiratory disease<br />
score (RDS) was assessed daily from 0 to 33 days after infection (Halbur et al., 1996).<br />
RDS scores could range from 0 to 6: 0 (no coughing), 1 (mild coughing after<br />
encouraged movement), 2 (mild coughing while leaving the pigs undisturbed), 3<br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
(moderate coughing after encouraged movement), 4 (moderate coughing while<br />
leaving the pigs undisturbed), 5 (severe coughing after encouraged movement), 6<br />
(severe coughing while leaving the pigs undisturbed). The number <strong>of</strong> coughing days<br />
was assessed as the total number <strong>of</strong> days on which at least one pig was coughing.<br />
Rectal temperatures were measured on a daily basis from 0-10 days after<br />
infection <strong>and</strong> every other day from 12-33 days after infection.<br />
To calculate average daily weight gain, the individual bodyweight <strong>of</strong> all<br />
animals was obtained on 0 days after infection, 7 days after infection, at the start <strong>of</strong><br />
the medication period (12 days after infection), 19 days after infection, 22 days after<br />
infection <strong>and</strong> 33 days after infection. To be able to calculate the feed intake <strong>and</strong> feed<br />
conversion ratio per treatment group, feed allocations were recorded daily from 0<br />
days after infection onwards throughout the duration <strong>of</strong> the study. Feed leftovers were<br />
removed <strong>and</strong> weighed on the mentioned pig weighing days.<br />
Post-mortem examinations<br />
All pigs were euthanased at 33 days after infection by deep anesthaesia with<br />
0.3 ml/kg <strong>of</strong> a mixture <strong>of</strong> xylazine (Xylm ® 2%; Intervet) <strong>and</strong> zolazepam <strong>and</strong> tiletamin<br />
(Zoletil ® 100; Virbac) followed by exsanguination. The lungs were removed <strong>and</strong><br />
macroscopic pneumonia lesions were quantified using a lung lesion score diagram<br />
(Hannan et al., 1982). Total lung lesion scores could vary between score 0 (no<br />
lesions) <strong>and</strong> a theoretical maximum <strong>of</strong> 35 (100% <strong>of</strong> the lung area affected by<br />
pneumonia). A digital picture (DSC-S50; Sony Cyber-shot) was taken from every<br />
lung for future reference.<br />
Two lung tissue samples were collected from every lung from sections with<br />
lesions typical for M. hyopneumoniae infection (preferably the cardiac lobes) or from<br />
healthy tissue when macroscopical lesions were not apparent. The 2 samples were<br />
processed in a semi-quantitative immun<strong>of</strong>luorescence assay (score 0-3) to detect the<br />
presence <strong>of</strong> M. hyopneumoniae (Kobisch et al., 1978); 0: (no immun<strong>of</strong>luorescence), 1,<br />
2 <strong>and</strong> 3 (limited, moderate <strong>and</strong> intense immun<strong>of</strong>luorescence, respectively). An<br />
additional lung tissue sample per pig was taken for bacteriological examination.<br />
Twenty percent (w/v) suspensions <strong>of</strong> lungs were made, inoculated on Columbia agar<br />
supplemented with 5 percent sheep blood (blood agar) <strong>and</strong> incubated at 37°C for 48<br />
hours to check the presence <strong>of</strong> secondary bacteria in the lungs. The plates were cross-<br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
streaked with Staphylococcus aureus for support <strong>of</strong> Actinobacillus pleuropneumoniae<br />
growth.<br />
To demonstrate the presence <strong>and</strong> localisation <strong>of</strong> tylosin in the lung, entire<br />
transverse sections from across the bottom <strong>of</strong> the diaphragmatic lobe <strong>of</strong> the right lung<br />
were collected. After sampling, the tissues were immersed in paraformaldehyde<br />
fixative for one month <strong>and</strong> sent to the Veterinary Laboratory Agency (VLA), New<br />
Haw, Addlestone, Surrey, UK for further processing. Each sample was cut into 4<br />
pieces <strong>and</strong> processed to paraffin wax embedded blocks. Immunostained sections were<br />
examined by light microscopy <strong>and</strong> subjectively assessed for specific cellular tylosin<br />
staining <strong>and</strong> diffuse non specific background staining. Digital photomicrographs<br />
(DX1200; Nikon Digital Camera, Nikon ACT-1 s<strong>of</strong>tware) <strong>of</strong> representative specific<br />
immunodetection in each experimental group were produced.<br />
Serology for M. hyopneumoniae<br />
A blood sample <strong>of</strong> every pig was taken at 0 <strong>and</strong> 33 days after infection to<br />
detect serum antibodies against M. hyopneumoniae, using the DAKO ® Mh ELISA<br />
(DAKO, Glostrup, Denmark) (Feld et al., 1992). Sera with optical density (OD)-<br />
values
2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
test was used to compare the number <strong>of</strong> animals between groups with a positive M.<br />
hyopneumoniae serology result <strong>and</strong> with a positive IF score. Statistical analyses were<br />
performed using SAS 8 (SAS Institute, 1999).<br />
RESULTS<br />
Clinical <strong>and</strong> performance parameters<br />
One animal in the NC group, two animals in the non-TP group <strong>and</strong> three<br />
animals in the TP group were found with arthritis, presumably caused by<br />
Streptococcus suis. They were intramuscularly injected 3 times with amoxycillin,<br />
every other day. One animal in the negative control group died during blood sampling<br />
on the day <strong>of</strong> infection. No other animals were removed from study.<br />
During the pre-medication period, coughing was rarely observed in the 3<br />
groups. For the entire study period, coughing was present in both groups inoculated<br />
with M. hyopneumoniae but was rarely observed in the NC group (Figure 1).<br />
Coughing started at approximately 11 days after infection <strong>and</strong> increased steadily<br />
towards the end <strong>of</strong> the study. Average RDS were significantly higher (P
2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
2.5<br />
2<br />
Average RDS<br />
1.5<br />
1<br />
0.5<br />
0<br />
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33<br />
DAI<br />
0 DAI<br />
infection<br />
13 DAI<br />
start tylosin treatment<br />
23 DAI<br />
33 DAI<br />
euthanasia<br />
pre-treatment period<br />
tylosin treatment period<br />
NC TP non-TP<br />
Figure 1: Average respiratory disease score (RDS) <strong>of</strong> the M. hyopneumoniae infected<br />
<strong>and</strong> tylosin treated group (TP) (n= 10 pigs), the M. hyopneumoniae infected <strong>and</strong> nontreated<br />
group (non-TP) (n= 10 pigs) <strong>and</strong> the non-infected <strong>and</strong> non-treated control<br />
(NC) group (n= 9 pigs), during the pre-treatment period (0-12 days after infection<br />
(DAI)) <strong>and</strong> the tylosin treatment period (13-33 DAI).<br />
The average rectal temperatures were not significantly different between the<br />
treatment groups (average temperatures were 39.5, 39.6 <strong>and</strong> 39.7 °C in the NC, non-<br />
TP <strong>and</strong> TP group, respectively). Rectal temperatures slowly decreased towards the<br />
end <strong>of</strong> the study in all groups.<br />
None <strong>of</strong> the performance parameters were significantly different between the<br />
study groups. The average weights (SD) for the NC, non-TP <strong>and</strong> TP group were 8.18<br />
(1.21), 8.07 (1.08) <strong>and</strong> 7.89 (1.42) kg at the beginning <strong>of</strong> the study, 8.85 (1.25), 9.70<br />
(1.38) <strong>and</strong> 9.42 (1.90) at 7 days after infection, 11.15 (1.55), 11.10 (1.54) <strong>and</strong> 11.10<br />
(2.30) at the start <strong>of</strong> the medication period (12 days after infection), 14.46 (1.95),<br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
15.44 (2.39) <strong>and</strong> 14.34 (3.36) at 19 days after infection, 16.38 (2.53), 17.14 (2.72) <strong>and</strong><br />
15.98 (3.74) at 22 days after infection <strong>and</strong> 23.87 (3.26), 23.19 (4.49) <strong>and</strong> 23.70 (5.05)<br />
kg at 33 days after infection, respectively. For the NC, non-TP <strong>and</strong> TP group, the<br />
ADG was 0.48 (0.08), 0.46 (0.11) <strong>and</strong> 0.48 (0.12) kg, the average daily feed intake<br />
per pig was 0.79, 0.80 <strong>and</strong> 0.78 kg <strong>and</strong> the FCR 1.64, 1.75 <strong>and</strong> 1.64, respectively.<br />
In the tylosin treatment group, the nominal intake <strong>of</strong> tylosin during the<br />
medication period was 5.7 mg/kg bodyweight, which was within the target intake<br />
range <strong>of</strong> 3-6 mg/kg bodyweight.<br />
Post-mortem examinations (Table 1)<br />
The proportion <strong>of</strong> animals with lung lesions was significantly higher (P
2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
A<br />
B<br />
Figure 2: Digital photographs <strong>of</strong> immunostained, paraffin embedded lung tissue<br />
sections, magnification x400. A: section <strong>of</strong> a pig <strong>of</strong> the non-infected, non-treated<br />
control group. B: section <strong>of</strong> a pig <strong>of</strong> the infected, tylosin treated group. Infiltrating<br />
macrophages show cytoplasmic tylosin (arrows).<br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
Serology for M. hyopneumoniae (Table 1)<br />
None <strong>of</strong> the 30 pigs had a positive ELISA result at 0 days after infection. The<br />
proportion <strong>of</strong> animals that seroconverted at 33 days after infection was significantly<br />
higher (P
2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
DISCUSSION<br />
In the swine industry, antibiotics are frequently used to prevent <strong>and</strong> treat<br />
diseases such as enzootic pneumonia, one <strong>of</strong> the most important respiratory diseases<br />
in swine worldwide. Outbreaks <strong>of</strong> enzootic pneumonia still occur frequently in<br />
intensive pig production systems <strong>and</strong> the onset cannot always be predicted. For those<br />
outbreaks, antibiotics are necessary to limit the number <strong>of</strong> diseased animals <strong>and</strong> the<br />
extent <strong>of</strong> the lesions <strong>and</strong> consequently to improve better animal welfare <strong>and</strong> limit the<br />
economical loss.<br />
Tylosin is one <strong>of</strong> the oldest antibiotics for veterinary medicine that is still<br />
frequently used (Chauvin et al., 2002). A previous study demonstrating the efficacy <strong>of</strong><br />
tylosin for the treatment <strong>of</strong> enzootic pneumonia was carried out with neonatal piglets<br />
inoculated intranasally with a lung homogenate containing different mycoplasma<br />
species. Treatment with tylosin tartrate was initiated at 14 days after infection <strong>and</strong><br />
lasted for 10 days (Hannan et al., 1982). In other studies, the preventive use <strong>of</strong> tylosin<br />
against M. hyopneumoniae infections was examined (Ueda et al., 1994). In our study,<br />
the efficacy <strong>of</strong> tylosin was evaluated at the onset <strong>of</strong> an enzootic pneumonia outbreak.<br />
Tylosin treatment only started at 12 days after infection, when approximately 10% <strong>of</strong><br />
the pigs were coughing. In this way, the study tried to simulate field conditions where<br />
a treatment is <strong>of</strong>ten initiated when 10-20% <strong>of</strong> the pigs are coughing.<br />
After oral treatment, tylosin has been shown to be widely distributed in pigs<br />
throughout body fluids <strong>and</strong> the drug also reaches peripheral tissues (Prats et al.,<br />
2002). In the present study, strong immunostaining was detected in the cytoplasm <strong>of</strong><br />
lung alveolar macrophages <strong>of</strong> pigs treated with tylosin, confirming that this antibiotic<br />
accumulates in these cells (Scorneaux <strong>and</strong> Shryock, 1998).<br />
Respiratory tract disease symptoms <strong>and</strong> lung lesions were less extensive <strong>and</strong><br />
less severe in the tylosin treated group than in the non-treated control group,<br />
demonstrating the efficacy <strong>of</strong> this antibiotic to treat enzootic pneumonia in swine<br />
inoculated with a high dose <strong>of</strong> a highly virulent M. hyopneumoniae isolate. The<br />
results <strong>of</strong> the semi-quantitative immun<strong>of</strong>luorescence indicated that tylosin treatment<br />
may decrease the number <strong>of</strong> M. hyopneumoniae organisms in the lungs, although<br />
average IF scores were not significantly different between the TP group <strong>and</strong> non-TP<br />
group. Kobisch et al., (1978) <strong>and</strong> Amanfu et al., (1984) showed that the intensity <strong>of</strong><br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
immun<strong>of</strong>luorescence has indeed been correlated with the number <strong>of</strong> organisms in the<br />
lungs. Secondary bacterial lung infections were only detected in 2 pigs <strong>of</strong> the nontreated<br />
positive control group. In severe field cases <strong>of</strong> enzootic pneumonia, the<br />
frequency <strong>of</strong> secondary bacterial infections is usually higher (Maes et al., 1998). The<br />
reason for this is not clear but may be due to different housing <strong>and</strong> management<br />
conditions (Clark et al., 1991). In our study, all pigs were simultaneously inoculated<br />
with a high dose <strong>of</strong> M. hyopneumoniae. In the field, the infection dose is not known<br />
but due to the chronic nature <strong>of</strong> the disease, repeated infections can be expected <strong>and</strong><br />
the infection <strong>of</strong> the pigs might be spread over time. Stocking density, ventilation <strong>and</strong><br />
hygienic measures may also differ substantially from the circumstances under<br />
experimental conditions. Nevertheless, experimental studies remain important to<br />
precisely evaluate the efficacy <strong>of</strong> antibiotics since in contrast with field studies,<br />
variables that are out <strong>of</strong> the control <strong>of</strong> the investigator are not likely to influence the<br />
results.<br />
CONCLUSION<br />
Tylosin administered via the feed at a level <strong>of</strong> 100 mg/kg feed for 21 days was<br />
efficacious for the treatment <strong>of</strong> established mycoplasmal disease. Even if M.<br />
hyopneumoniae vaccination is widely used in modern pig herds, it remains important<br />
to have effective, easily applicable antibiotics which can be used at the moment <strong>of</strong><br />
acute outbreaks <strong>of</strong> enzootic pneumonia.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by a grant from Elanco Animal <strong>Health</strong> Benelux, a<br />
Divison <strong>of</strong> Eli Lilly <strong>and</strong> Co.<br />
The authors are grateful to Mrs. Y. Spencer from the VLA, New Haw,<br />
Addlestone, Surrey, UK, for providing the figures demonstrating the presence <strong>of</strong><br />
tylosin in the alveolar macrophages.<br />
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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
REFERENCES<br />
1. Amanfu, W., Weng, C., Ross, R.F. & Barnes, H. (1984) Diagnosis <strong>of</strong> mycoplasmal<br />
pneumonia <strong>of</strong> swine: sequential study by direct immun<strong>of</strong>luorescence. American Journal <strong>of</strong><br />
Veterinary Research 45, 1349-1352.<br />
2. Brisson-Noël, A., Trieu-Cuot, P. & Courvalin, P. (1988) Mechanism <strong>of</strong> action <strong>of</strong> spiramycin<br />
<strong>and</strong> other macrolides. The Journal <strong>of</strong> Antimicrobial Chemotherapy 22, 13.23.<br />
3. Chauvin, C., Beloeil, P-A., Or<strong>and</strong>, J-P., S<strong>and</strong>ers, P. & Madec, F. (2002) A survey <strong>of</strong> grouplevel<br />
antibiotic prescriptions in pig production in France. Preventive Veterinary Medicine 55,<br />
109-120.<br />
4. Clark, L., Armstrong, C., Knox, K. & Mayrose, V. (1991) The effect <strong>of</strong> all-in/all-out<br />
management on pigs from a herd with enzootic pneumonia. Veterinary Medicine 86, 946-951.<br />
5. Dohoo, I., & Montgomery, M. (1996) A field trial to evaluate a Mycoplasma hyopneumoniae<br />
vaccine: effects on lung lesions <strong>and</strong> growth rates in swine. Canadian Veterinary Journal 37, 299-<br />
302.<br />
6. Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F. & Meyling, A. (1992) A monoclonal blocking<br />
ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. Veterinary Microbiology 30,<br />
35-46.<br />
7. Halbur, P.G., Paul, P.S., Meng, X.J., Lum, M.A., Andrews, J.J. & Rathje, J.A. (1996)<br />
Comparative pathogenicity <strong>of</strong> nine US Porcine Reproductive <strong>and</strong> Respiratory Syndrome Virus<br />
(PRRSV) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model. Journal <strong>of</strong><br />
Veterinary Diagnostic Investigation 8, 11-20.<br />
8. Hannan, P.C., Bhogal, B.S. & Fish, J.P. (1982) Tylosin tartrate <strong>and</strong> tiamutilin effects on<br />
experimental piglet pneumonia induced with pneumonic pig lung homogenate containing<br />
mycoplasmas, bacteria <strong>and</strong> viruses. Research in Veterinary Science 33, 76-88.<br />
9. Kobisch, M., Tillon, J.P., Vannier, Ph., Magueur, S. & Morvan, P. (1978) Pneumonie<br />
enzootique à Mycoplasma suipneumoniae chez le porc: diagnostic rapide et recherche<br />
d’anticorps. Récueil de Médecine Vétérinaire 154, 847-852.<br />
10. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Lein, A., Vrijens, B. & de<br />
Kruif, A. (1998) Effect <strong>of</strong> vaccination against Mycoplasma hyopneumoniae in pig herds with a<br />
continuous production system. Journal <strong>of</strong> Veterinary Medicine B 45, 495-505.<br />
11. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A.<br />
(1999a) Risk indicators for the seroprevalence <strong>of</strong> Mycoplasma hyopneumoniae, porcine<br />
influenza viruses <strong>and</strong> Aujeszky’s disease virus in slaughter pigs from fattening pig herds.<br />
Journal <strong>of</strong> Veterinary Medicine B 46, 341-352.<br />
12. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., Verbeke, W.,<br />
Viaene, J. & de Kruif, A. (1999b) Effect <strong>of</strong> vaccination against Mycoplasma hyopneumoniae in<br />
pig herds with an all-in/all-out production system. Vaccine 17, 1024-34.<br />
13. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A.<br />
(2000) <strong>Herd</strong> factors associated with the seroprevalences <strong>of</strong> four major respiratory pathogens in<br />
slaughter pigs from farrow-to-finish pig herds. Veterinary Research 31, 313-327.<br />
14. Prats, C., El Korchi, G., Francesch, R., Arboix, M. & Pérez, B. (2002) Disposition kinetics<br />
<strong>of</strong> tylosin administered intravenously <strong>and</strong> intramuscularly to pigs. Research in Veterinary<br />
Science 73, 141-144.<br />
- 172 -
2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS<br />
15. SAS Institute, Statistical Analysis Systems (1999) SAS/STAT user’s guide, Version 8. SAS<br />
Institute Inc., Cary, NC, USA<br />
16. Scorneaux, B. & Shryock, T.R. (1998) Intracellular accumulation, subcellular distribution <strong>and</strong><br />
efflux <strong>of</strong> tilmicosin in swine phagocytes. Journal <strong>of</strong> Veterinary Pharmacology <strong>and</strong> Therapeutics<br />
21, 257-268.<br />
17. Tanner, A.C., Erickson, B.Z. & Ross, R.F. (1993) Adaptation <strong>of</strong> the Sensititre ® broth<br />
microdilution technique to antimicrobial susceptibility testing <strong>of</strong> Mycoplasma hyopneumoniae.<br />
Veterinary Microbiology 36, 301-306.<br />
18. Ter Laak, E.A., Pijpers, A., Noordergraaf, J.H., Schoevers, E.C. & Verheijden, J.H.M.<br />
(1991) Comparison <strong>of</strong> methods for in vitro testing <strong>of</strong> susceptibility <strong>of</strong> porcine mycoplasma<br />
species to antimicrobial agents. Antimicrobial Agents <strong>and</strong> Chemotherapy 35, 228-233.<br />
19. Ueda, Y., Oktsuki, S., Narukawa, N., Takeda K. (1994) Effect <strong>of</strong> terdecamycin on<br />
experimentally induced Mycoplasma hyopneumoniae-infection in pigs. Journal <strong>of</strong> Veterinary<br />
Medicine, Series B 41, 283-290.<br />
20. Vicca, J., Stakenborg, T., Maes, D., Butaye, P., de Kruif, A. & Haesebrouck, F. (2002)<br />
Antibiotic susceptibility <strong>of</strong> Belgian Mycoplasma hyopneumoniae field isolates. Proceedings <strong>of</strong><br />
the 14th international congress <strong>of</strong> the International Organisation for Mycoplasmology; Vienna,<br />
Austria, 61-62.<br />
21. Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. & Haesebrouck, F.<br />
(2003) Evaluation <strong>of</strong> virulence <strong>of</strong> Mycoplasma hyopneumoniae field isolates. Veterinary<br />
Microbiology 97, 177-190.<br />
22. Wu, C.C., Shryock, T.R., Lin, T.L. & Veenhuizen, M.F. (1997) Testing antimicrobial<br />
susceptibility against Mycoplasma hyopneumoniae in vitro. Journal <strong>of</strong> Swine <strong>Health</strong> <strong>and</strong><br />
Production 5, 227-230.<br />
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3 GENERAL DISCUSSION
3 GENERAL DISCUSSION<br />
Infections with M. hyopneumoniae, the primary agent <strong>of</strong> enzootic pneumonia<br />
in pigs, are very common worldwide, especially in countries with intensive<br />
production systems. Enzootic pneumonia causes major economic losses to the pig<br />
industry due to decreased daily weight gain, increased feed conversion ratio, number<br />
<strong>of</strong> days to reach slaughter weight <strong>and</strong> medication costs. Losses in the Belgian pig<br />
industry are estimated to be at least 14 million euro each year. Studying the factors<br />
influencing the disease-course, which may evolve either subclinically or clinically, is<br />
essential to limit or reduce these losses. The best studied factor is the influence <strong>of</strong> the<br />
environment on the disease outcome. Poor management <strong>and</strong> housing conditions have<br />
been considered to be important deteriorating factors (Clark et al., 1991; Maes et al.,<br />
1996; Ross, 1999). The host susceptibility, in contrast, is not found to be a major<br />
contributing factor (Goodwin et al., 1969; Piffer <strong>and</strong> Ross, 1984; Kobisch et al., 1993;<br />
Wallgren et al., 1998). The third factor that may influence the disease-course is<br />
related to the organism causing the disease. Antigenic <strong>and</strong> genetic diversity <strong>of</strong> M.<br />
hyopneumoniae isolates has been reported earlier (Ro <strong>and</strong> Ross, 1983; Frey et al.,<br />
1992; Artiushin <strong>and</strong> Minion, 1996) but no correlation was made with the virulence. In<br />
this thesis, differences in virulence between M. hyopneumoniae field isolates were<br />
studied.<br />
In a first field study (chapter 2.1.1), herds with either poor housing <strong>and</strong><br />
management conditions but experiencing subclinical disease or herds with good<br />
housing <strong>and</strong> management conditions but with a clinical disease-course were selected.<br />
This selection procedure was used to increase the chance <strong>of</strong> isolating low <strong>and</strong> high<br />
virulence M. hyopneumoniae isolates.<br />
Six isolates obtained in the first study were used in an experimental infection<br />
model (chapter 2.1.2) to asses the virulence. This model proved to be highly<br />
reproducible <strong>and</strong> can be used to evaluate the efficacy <strong>of</strong> vaccins or antimicrobial<br />
treatment. It also allows classifying the isolates as low, moderately <strong>and</strong> highly<br />
virulent.<br />
Animals inoculated with a high virulence isolate developed more severe lung<br />
lesions. In the lungs <strong>of</strong> these animals, the immun<strong>of</strong>luorescence score was significantly<br />
higher than in the lungs <strong>of</strong> animals inoculated with a low virulence strain. This<br />
indicates that a higher number <strong>of</strong> M. hyopneumoniae organisms in the lungs results in<br />
more severe lung lesions. The higher immun<strong>of</strong>luorescence score can be due to a faster<br />
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3 GENERAL DISCUSSION<br />
multiplication in the host, a better circumvention or modulation <strong>of</strong> the host immune<br />
response or a more efficient adherence <strong>of</strong> high virulence isolates. A difference in<br />
capability to attach to cilia between M. hyopneumoniae strains has been demonstrated<br />
by Minion et al. (2000) <strong>and</strong> was correlated with the number <strong>of</strong> repeat regions in RR1<br />
<strong>of</strong> the p97 protein <strong>of</strong> M. hyopneumoniae. In that study, a minimum <strong>of</strong> eight repeats<br />
was necessary to make the M. hyopneumoniae organisms able to attach to the cilia.<br />
Recently, a difference in the number <strong>of</strong> repeats in RR1 was detected in our isolates (T.<br />
Stakenborg, personal communication, 2005) but no correlation was found between the<br />
number <strong>of</strong> repeats <strong>and</strong> the virulence <strong>of</strong> the isolates.<br />
Using the OPA-3 primer (5’ AGTCAGCCAC 3’) (Artiushin <strong>and</strong> Minion,<br />
1996), a RAPD b<strong>and</strong> <strong>of</strong> about 5 kbp was only found in high <strong>and</strong> moderate virulence<br />
isolates, but not in low virulence isolates. In further studies, this RAPD b<strong>and</strong> was<br />
sequenced (accession n° AY551928) <strong>and</strong> shown to be 5319 bp in size. Specific<br />
primers were developed for amplification <strong>of</strong> this 5319 bp fragment. PCR with these<br />
primers resulted in amplification <strong>of</strong> the 5319 bp fragment in all M. hyopneumoniae<br />
isolates tested. Sequencing <strong>of</strong> the amplicons obtained in high, moderate <strong>and</strong> low<br />
virulence isolates revealed only minor differences in the OPA-3 binding sites. The<br />
observed differences were not consistent with the virulence <strong>of</strong> the isolates. It remains<br />
unclear why RAPD with the OPA-3 primers did not result in a 5 kbp b<strong>and</strong> in low<br />
virulence isolates. Possibly, some smaller b<strong>and</strong>s were preferentially amplified in low<br />
virulence isolates compared to isolates <strong>of</strong> higher virulence, inhibiting amplification <strong>of</strong><br />
the 5 kbp b<strong>and</strong> during RAPD analyses. It can be concluded that although RAPD with<br />
OPA-3 might be useful to predict virulence <strong>of</strong> M. hyopneumoniae isolates, it is clear<br />
that the b<strong>and</strong> itself does not carry any gene that may explain the differences in<br />
virulence <strong>of</strong> isolates observed in vivo.<br />
The fact that clear differences in virulence between isolates were observed<br />
may have important consequences at herd level. It is likely that herds infected with<br />
high virulence M. hyopneumoniae isolates will experience considerably higher<br />
economical losses than herds infected with low virulence isolates. A diagnostic test<br />
discriminating high <strong>and</strong> low virulence isolates would be very useful to implement the<br />
most appropriate control strategies.<br />
The availability <strong>of</strong> a collection <strong>of</strong> M. hyopneumoniae isolates <strong>of</strong> well<br />
determined virulence creates various possibilities for further research. Using our<br />
strains, Meyns et al. (2004) quantified the spread <strong>of</strong> high <strong>and</strong> low virulence isolates.<br />
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3 GENERAL DISCUSSION<br />
Although a faster spread <strong>of</strong> high virulence isolates was observed, the adjusted<br />
reproduction ratio (R n ) values were not significantly different. Macrophages are found<br />
to be the dominant cells present in lungs infected with M. hyopneumoniae (Sarradell<br />
et al., 2003) <strong>and</strong> pro-inflammatory cytokines which are mainly produced by<br />
macrophages play a prominent role in the lesion development (Henderson et al.,<br />
1996). It would not be inconceivable that infections with high virulence isolates result<br />
in higher production <strong>of</strong> pro-inflammatory cytokines by macrophages compared to<br />
infections with low virulence isolates. A difference in macrophage stimulation was<br />
observed by Jungi et al. (1996) comparing different strains <strong>of</strong> Mycoplasma mycoides<br />
ssp. mycoides small colony type. In Mycoplasma fermentans, a lipoprotein called<br />
macrophage activating lipoprotein 2 kDa (MALP-2), was characterized <strong>and</strong> found to<br />
be responsible for the induction <strong>of</strong> pro-inflammatory cytokines (Seya <strong>and</strong> Matsumoto,<br />
2002). Further studies on lipoproteins present in M. hyopneumoniae might provide<br />
more insights in the pathogenesis <strong>of</strong> enzootic pneumonia.<br />
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3 GENERAL DISCUSSION<br />
The second part <strong>of</strong> this PhD deals with in vitro <strong>and</strong> in vivo susceptibility <strong>of</strong> M.<br />
hyopneumoniae isolates to some groups <strong>of</strong> antimicrobials. This information is<br />
essential to avoid ineffective treatments. Since M. hyopneumoniae is a very fastidious<br />
organism that is difficult to isolate <strong>and</strong> cultivate, data on the prevalence <strong>of</strong> acquired<br />
resistance are very scarce in the literature. Also, susceptibility information from other<br />
countries may be misleading since the type <strong>of</strong> antimicrobials <strong>and</strong> the amount<br />
administered may differ substantially due to country regulations in antimicrobial use,<br />
eg. fluoroquinolones are not allowed for use in pigs in the US but are allowed in the<br />
EU (World <strong>Health</strong> Organization, 1998; American Veterinarian Medical Association,<br />
2005).<br />
Based on the literature (Hannan et al., 1989; Ter Laak et al., 1991; Tanner et<br />
al., 1993; Friis <strong>and</strong> Szancer, 1994; Hannan et al., 1997), a high prevalence <strong>of</strong> isolates<br />
with acquired resistance was not expected. Only Inamoto et al. (1994) found several<br />
M. hyopneumoniae isolates with decreased susceptibility to oxytetracycline. In our<br />
studies, acquired resistance to tetracyclines was not detected in spite <strong>of</strong> the frequent<br />
use <strong>of</strong> these antibiotics in swine herds. One <strong>of</strong> the 21 Belgian M. hyopneumoniae<br />
isolates tested was found to have decreased susceptibility to macrolides <strong>and</strong><br />
lincosamides, while in 5 isolates the susceptibility towards fluoroquinolones was<br />
decreased. Macrolides <strong>and</strong> lincosamides are frequently used for the treatment <strong>of</strong> M.<br />
hyopneumoniae infections in Western Europe, while fluoroquinolones are only<br />
occasionally used (Chauvin et al., 2002; Timmerman et al., 2005).<br />
Acquired resistance to an antimicrobial is the result <strong>of</strong> an alteration <strong>of</strong> the<br />
genome <strong>of</strong> a microorganism. It may arise because <strong>of</strong> a mutation in a gene or through<br />
horizontal transfer <strong>of</strong> resistance genes. Transfer-resistance has been described in M.<br />
pulmonis (Teachman et al., 2002) but no other reports on horizontal transfer <strong>of</strong><br />
resistance genes exist. In chapter 2.2.2, it was shown that resistance to macrolides <strong>and</strong><br />
lincosamides in our M. hyopneumoniae isolate was due to a A2058G mutation in the<br />
23S rRNA gene. A C264A mutation in parC <strong>and</strong> a C635T mutation in gyrA were<br />
found to be responsible for decreased susceptibility to enr<strong>of</strong>loxacin <strong>and</strong> flumequine<br />
(chapter 2.2.3). These mutations result in a modification <strong>of</strong> the target site for these<br />
antimicrobials. Resistance against fluoroquinolones might also be efflux mediated as<br />
has been described in M. hominis where reduced cipr<strong>of</strong>loxacin accumulation was<br />
related to the overexpression <strong>of</strong> two genes encoding a putative ABC type efflux<br />
(Raherison et al., 2002). However, it has been demonstrated that mutations in genes<br />
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3 GENERAL DISCUSSION<br />
encoding the target sites <strong>of</strong> fluoroquinolones are the major cause <strong>of</strong> the increase in<br />
MIC <strong>and</strong> efflux mediated resistance is only supplementary (Poole, 2005). Whether<br />
efflux-mechanisms contribute to the increased MIC-values <strong>of</strong> fluoroquinolones for M.<br />
hyopneumoniae needs further investigation.<br />
Several factors influence the development <strong>and</strong> spread <strong>of</strong> antimicrobial<br />
resistant micro-organisms. The antimicrobial use is one factor gaining much attention.<br />
The selection pressure exerted by antimicrobials is influenced by the drug regimen,<br />
which includes the dose, the treatment interval, the duration <strong>of</strong> treatment <strong>and</strong> the<br />
formulation (Catry et al., 2003). A theoretical explanation <strong>of</strong> the influence <strong>of</strong> these<br />
factors on the development <strong>of</strong> de novo resistance is the “mutant selection window”<br />
(Catry et al., 2003; Drlica, 2003; Epstein et al., 2004). The mutant selection window<br />
is an antimicrobial concentration range extending from the MIC 99 to the mutant<br />
prevention concentration (MPC) which can be defined as the antimicrobial<br />
concentration required for blocking the growth <strong>of</strong> the least susceptible, single-step<br />
mutant. Antimicrobial concentrations fitted within this window enrich subpopulations<br />
selectively. Traditional dosing recommendations to exceed MICs are likely to place<br />
drug concentrations inside the selection window. In our study (chapter 2.2.1), the use<br />
<strong>of</strong> enr<strong>of</strong>loxacin in suckling piglets was striking. This drug was at least used in the<br />
farrowing unit on all herds where a resistant isolate was obtained. Delsol et al. (2004<br />
a;b) demonstrated that administration <strong>of</strong> registered doses <strong>of</strong> enr<strong>of</strong>loxacin for 5 days to<br />
weaned piglets selected for enr<strong>of</strong>loxacin resistant Campylobacter coli isolates <strong>and</strong> for<br />
quinolone (but not fluoroquinolone) resistant Salmonella enterica serovar<br />
Typhimurium isolates. The use <strong>of</strong> enr<strong>of</strong>loxacin in suckling piglets may have selected<br />
low level resistant strains due to a mutation in parC. This low level resistant<br />
population may have been sustained on the herd by the use <strong>of</strong> a drug dose within the<br />
mutant selection window possibly resulting in selection <strong>of</strong> a population with higher<br />
level resistance on one herd due to an additional mutation in gyrA. The same<br />
hypothesis could be raised concerning the use <strong>of</strong> lincomycin which on one herd may<br />
have resulted in spread <strong>of</strong> mutants in the 23S rRNA gene. Experimental studies<br />
designed to substantiate this hypothesis may provide practical evidence for future<br />
treatment regimens.<br />
In a final study, our experimental infection model was used to evaluate in vivo<br />
effectiveness <strong>of</strong> tylosin (chapter 2.2.4). It was shown that this antimicrobial,<br />
belonging to the 16-membered macrolides, can be used to limit the extent <strong>of</strong><br />
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3 GENERAL DISCUSSION<br />
macroscopic lung lesions <strong>and</strong> consequently to reduce the clinical signs. Macrolide<br />
antibiotics accumulate in many tissues such as the epithelial lining fluid <strong>and</strong> it has<br />
been shown that the macrolide concentration in respiratory tract tissues <strong>and</strong><br />
extracellular fluids is higher than simultaneously measured serum concentrations (Jain<br />
<strong>and</strong> Danzinger, 2004). Results <strong>of</strong> immun<strong>of</strong>luorescence studies demonstrated that<br />
although tylosin treatment is able to reduce the number <strong>of</strong> M. hyopneumoniae<br />
organisms in the lungs, not all bacteria are eliminated. The reason for the latter<br />
finding is not clear but narrowing <strong>of</strong> small blood vessels in the lungs due to<br />
accumulation <strong>of</strong> inflammatory cells around small airways <strong>and</strong> blood vessels may play<br />
a role. Diffusion <strong>of</strong> the drug from the blood to the lung tissue might be delayed or<br />
hampered resulting in diposition <strong>of</strong> the drug mainly at places with lower numbers <strong>of</strong><br />
organisms. Additionally, the location <strong>of</strong> M. hyopneumoniae on the apex <strong>of</strong> the cilia<br />
<strong>and</strong> microvilli may also be a disadvantage for the accessiblity by the drug.<br />
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3 GENERAL DISCUSSION<br />
REFERENCES<br />
1. American Veterinary Medical Association. (2005). Judicious use <strong>of</strong> antimicrobials for swine<br />
veterinarians (Online). http://www.avma.org/scienact/jtua/swine/jtuaswine.asp.<br />
2. Artiushin, S. & Minion, F.C. (1996). Arbitrarily primed PCR analysis <strong>of</strong> Mycoplasma<br />
hyopneumoniae field isolates demonstrates genetic heterogeneity. International Journal <strong>of</strong><br />
Systemic Bacteriology 46, 324-328.<br />
3. Catry, B., Laevens, H., Devriese, L.A., Opsomer, G. & de Kruif, A. (2003). Antimicrobial<br />
resistance in livestock. Journal <strong>of</strong> Veterinary Pharmacology <strong>and</strong> Therapy 26, 81-93.<br />
4. Chauvin, C., Beloeil, P-A., Or<strong>and</strong>, J-P., S<strong>and</strong>ers, P. & Madec, F. (2002). A survey <strong>of</strong> grouplevel<br />
antibiotic prescriptions in pig production in France. Preventive Veterinary Medicine<br />
55,109-120.<br />
5. Clark, L., Armstrong, C., Knox, K. & Mayrose, V. (1991). The effect <strong>of</strong> all-in/all-out<br />
management on pigs from a herd with enzootic pneumonia. Veterinary Medicine 86, 946-951.<br />
6. Delsol, A.A., Sunderl<strong>and</strong>, J., Woodward, M.J., Pumbwe, L., Piddock, L.J. & Roe, J.M.<br />
(2004b). Emergence <strong>of</strong> fluoroquinolone resistance in the native Campylobacter coli population<br />
<strong>of</strong> pigs exposed to enr<strong>of</strong>loxacin. Journal <strong>of</strong> Antimicrobial Chemotherapy 53, 872-874.<br />
7. Delsol, A.A., Woodward, M.J. & Roe, J.M. (2004a). Effect <strong>of</strong> a 5 day enr<strong>of</strong>loxacin treatment<br />
on Salmonella enterica serotype Typhimurium DT104 in the pig. Journal <strong>of</strong> Antimicrobial<br />
Chemotherapy 53, 396-398.<br />
8. Drlica, K. (2003). The mutant selection window <strong>and</strong> antimicrobial resistance. Journal <strong>of</strong><br />
Antimicrobial Chemotherapy 52, 11-17.<br />
9. Epstein, B.J., Gums, J.G. & Drlica, K. (2004). The changing face <strong>of</strong> antibiotic prescribing: the<br />
mutant selection window. The Annals <strong>of</strong> Pharmacotherapy, 38, 1675-1682.<br />
10. Frey, J., Haldimann, A. & Nicolet, J. (1992). Chromosomal heterogeneity <strong>of</strong> various<br />
Mycoplasma hyopneumoniae field strains. International Journal <strong>of</strong> Systemic Bacteriology 42,<br />
275-280.<br />
11. Friis, N.F. & Szancer, J. (1994). Sensitivity <strong>of</strong> certain porcine <strong>and</strong> bovine mycoplasmas to<br />
antimicrobial agents in a liquid medium test compared to a disc assay. Acta Veterinaria<br />
Sc<strong>and</strong>inavia 35, 389-394.<br />
12. Goodwin, R., Hodgson, R., Wittlestone, P. & Woodhams, R. (1969). Immunity in<br />
experimentally induced enzootic pneumonia <strong>of</strong> pigs. Journal <strong>of</strong> Hygiene (London) 67, 193-208.<br />
13. Hannan, P.C.T., O’Hanlon, P.J. & Rogers, N.H. (1989). In vitro evaluation <strong>of</strong> various<br />
quinolone antibacterial agents against veterinary mycoplasmas <strong>and</strong> porcine respiratory bacterial<br />
pathogens. Research in Veterinary Science 46, 202-211.<br />
14. Hannan, P.C.T., Windsor, H.M. & Ripley, P.H. (1997). In vitro susceptibilities <strong>of</strong> recent field<br />
isolates <strong>of</strong> Mycoplasma hyopneumoniae <strong>and</strong> Mycoplasma hyopsynoviae to valnemulin<br />
(Econor ® ), tiamulin <strong>and</strong> enr<strong>of</strong>loxacin <strong>and</strong> the in vitro development <strong>of</strong> resistance to certain<br />
antimicrobial agents in Mycoplasma hyopneumoniae. Research in Veterinary Science 63, 157-<br />
160.<br />
15. Henderson, B., Poole, S. & Wilson, M. (1996). Bacterial modulins: a novel class <strong>of</strong> virulence<br />
factors which cause host tissue pathology by inducing cytokine synthesis. Microbiological<br />
re<strong>view</strong>s 60, 316-341.<br />
- 183 -
3 GENERAL DISCUSSION<br />
16. Inamoto, T., Takahashi, H., Yamamoto, K., Nakai, Y. & Ogimoto, K. (1994). Antibiotic<br />
susceptibility <strong>of</strong> Mycoplasma hyopneumoniae isolated from swine. Journal <strong>of</strong> Veterinary<br />
Medical Science 56, 393-394.<br />
17. Jain, R. & Danzinger, L.H. (2004). The macrolide antibiotics: a pharmacokinetic <strong>and</strong><br />
pharmacodinamic over<strong>view</strong>. Current Pharmaceutical Design 10, 3045-3053.<br />
18. Jungi, T.W., Krampe, M., Sileghem, M., Griot, C. & Nicolet, J. (1996). Differential <strong>and</strong><br />
strain-specific triggering <strong>of</strong> bovine alveolar macrophage effector functions by mycoplasmas.<br />
Microbial Pathogenesis 21, 487-498.<br />
19. Kobisch, M., Blanchard, B., Portier, M. & Le Potier, M. (1993). Mycoplasma<br />
hyopneumoniae infection in pigs: duration <strong>of</strong> the disease <strong>and</strong> resistance to reinfection.<br />
Veterinary Research 24, 67-77.<br />
20. Maes, D., Verdonck, M., Deluyker, H., de Kruif, A. (1996). Enzootic pneumonia in pigs: a<br />
re<strong>view</strong>. The Veterinary Quarterly 18, 104-109.<br />
21. Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F. & de Kruif, A. (2004).<br />
Quantification <strong>of</strong> the spread <strong>of</strong> Mycoplasma hyopneumoniae in nursery pigs using transmission<br />
experiments. Preventive Veterinary Medicine 66, 265-275.<br />
22. Minion, F.C., Adams, C. & Hsu, T. (2000). R& region <strong>of</strong> P97 mediates adherence <strong>of</strong><br />
Mycoplasma hyopneumoniae to swine cilia. Infection <strong>and</strong> Immunity 68, 3056-3060.<br />
23. Negri, M.C., Lipsitch, M., Blazquez, J., Levin, B.R. & Baquero, F. (2000). Concentrationdependent<br />
selection <strong>of</strong> small phenotypic difference in TEM β-lactamase-mediated antibiotic<br />
resistance. Antimicrobial Agents <strong>and</strong> Chemotherapy 44, 2485-2491.<br />
24. Piffer, I. & Ross, R. (1984). Effect <strong>of</strong> age on susceptibility <strong>of</strong> pigs to Mycoplasma<br />
hyopneumoniae pneumonia. American Journal <strong>of</strong> Veterinary Research 45, 478-481.<br />
25. Poole, K. (2005). Efflux-mediated antimicrobial resistance. Journal <strong>of</strong> Antimicrobial<br />
Chemotherapy 56, 20-51.<br />
26. Raherison, S., Gonzales, P., Renaudin, H., Charron, A., Bébéar, C. & Bébéar, C.M. 2002.<br />
Evidence <strong>of</strong> active efflux in resistance to cipr<strong>of</strong>loxacin <strong>and</strong> to ethidium bromide by Mycoplasma<br />
hominis. Antimicrobial Agents <strong>and</strong> Chemotherapy, 46, 672-679.<br />
27. Ro, L. & Ross, R.F. (1983). Comparison <strong>of</strong> Mycoplasma hyopneumoniae strains by serologic<br />
methods. American Journal <strong>of</strong> Veterinary Research 44, 2087-2094.<br />
28. Ross, R.F. (1999): Mycoplasmal diseases. In: Leman A.D., Straw B.E., Mengeling W.L.,<br />
Allaire S.D., Taylor D.J (Editors), Diseases <strong>of</strong> Swine, 8th Edition, Iowa State University Press,<br />
Ames, pp. 495-509.<br />
29. Sarradell, J., Andrada, M., Ramírez, A.S., Fernández, A., Gómez-Villam<strong>and</strong>os, J.C., Jover,<br />
A., Lorenzo, H., Herráez, P. & Rodríguez, F. (2003). A morphological <strong>and</strong><br />
immunohistochemical study <strong>of</strong> the bronchus-associated lymphoid tissue <strong>of</strong> pigs naturally<br />
infected with Mycoplasma hyopneumoniae. Veterinary Pathology 40, 395-404.<br />
30. Seya, T. & Matsumoto, M. (2002). A lipoprotein family from Mycoplasma fermentans confers<br />
host immune activation through Toll-like receptor 2. The International Journal <strong>of</strong> Biochemistry<br />
& Cell Biology 34, 901-906.<br />
31. Tanner, A.C., Erickson, B.Z. & Ross, R.F. (1993). Adaptation <strong>of</strong> the Sensititre ® broth<br />
microdilution technique to antimicrobial susceptibility testing <strong>of</strong> Mycoplasma hyopneumoniae.<br />
Veterinary Microbiology 36, 301-306.<br />
- 184 -
3 GENERAL DISCUSSION<br />
32. Ter Laak, E.A., Pijpers, A., Noordergraaf, J.H., Schoevers, E.C. & Verheijden, J.H.M.<br />
(1991). Comparison <strong>of</strong> methods for in vitro testing <strong>of</strong> susceptibility <strong>of</strong> porcine Mycoplasma<br />
species to antimicrobial agents. Antimicrobial Agents <strong>and</strong> Chemotherapy 35, 228-233.<br />
33. Teachman, A.M., French, C.T., Yu, H., Simmons, W.L. & Dybvig, K. (2002). Gene transfer<br />
in Mycoplasma pulmonis. Journal <strong>of</strong> Bacteriology 184, 947-951.<br />
34. Timmerman T., Dewulf J., Catry B., Feyen B., Opsomer G., de Kruif A. & Maes D. (2005).<br />
Quantification <strong>and</strong> evaluation <strong>of</strong> antimicrobial-drug use in group treatments for fattening pigs in<br />
Belgium. Preventive Veterinary Medicine, accepted.<br />
35. Wallgren, O., Bolske, G., Gustafsson, S., Mattsson, S., Fossum, C. (1998). Humoral immune<br />
responses to Mycoplasma hyopneumoniae in sows <strong>and</strong> <strong>of</strong>fspring following an outbreak <strong>of</strong><br />
mycoplasmosis. Veterinary Microbiology 60, 193-205.<br />
36. World <strong>Health</strong> Organization. (1998). Use <strong>of</strong> quinolones in food animals <strong>and</strong> potential impact<br />
on human health. (Online). http:www.who.int/emcdocuments/zoonoses/docs/whoemczdi9810.html#Quinolone%20use%20in%animals.<br />
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4 SUMMARY
4 SUMMARY<br />
Mycoplasma hyopneumoniae is the primary cause <strong>of</strong> enzootic pneumonia in<br />
pigs, a chronic disease which is responsible for major economic losses in the pig<br />
industry worldwide. The mean factors influencing the disease-course <strong>and</strong> economic<br />
losses mentioned in the literature are environmental factors <strong>and</strong> herd management.<br />
A re<strong>view</strong> <strong>of</strong> the literature focussing on the pathogenesis <strong>of</strong> enzootic<br />
pneumonia is given in chapter 1.1.1 <strong>of</strong> this PhD thesis. In chapter 1.1.2, an over<strong>view</strong><br />
<strong>of</strong> antimicrobial agents <strong>and</strong> their in vitro <strong>and</strong> in vivo activity against M.<br />
hyopneumoniae is presented. Development <strong>of</strong> resistance in Mycoplasma spp. to<br />
circumvent the antibiotic action <strong>and</strong> the related resistance mechanisms are<br />
summarized. Control <strong>of</strong> enzootic pneumonia can be achieved using preventive<br />
medication or vaccination. Advantages <strong>and</strong> disadvantages <strong>of</strong> both strategies are<br />
discussed. Finally, M. hyopneumoniae eradication strategies developed <strong>and</strong> applied in<br />
some countries are described.<br />
The general aims <strong>of</strong> this thesis were to evaluate the virulence <strong>of</strong> M.<br />
hyopneumoniae field isolates <strong>and</strong> to study their antimicrobial susceptibility.<br />
In chapter 2.1.1, the patterns <strong>of</strong> M. hyopneumoniae infections were<br />
investigated in five herds with clinical evidence <strong>of</strong> enzootic pneumonia <strong>and</strong> in five<br />
herds subclinically infected with M. hyopneumoniae. In the clinically infected herds,<br />
housing <strong>and</strong> management conditions were good whereas these conditions were poor<br />
in the subclinically infected herds. In each herd, serum antibodies against M.<br />
hyopneumoniae were determined in pigs <strong>of</strong> different ages <strong>and</strong> nasal swabs were taken<br />
for M. hyopneumoniae detection using nested PCR (nPCR). The percentage <strong>of</strong><br />
seropositive pigs in the clinically infected herds increased from 8% in pigs <strong>of</strong> 9 weeks<br />
to 52% in pigs <strong>of</strong> 18 weeks <strong>and</strong> seroconversion was most frequently observed<br />
between 12 <strong>and</strong> 15 weeks <strong>of</strong> age. In the subclinically infected herds, the percentages<br />
increased from 2 to 24% <strong>and</strong> most <strong>of</strong> the pigs became seropositive between 15 <strong>and</strong> 18<br />
weeks. The percentage <strong>of</strong> nPCR positive pigs at 6 weeks was 16% <strong>and</strong> 0% in the<br />
clinically <strong>and</strong> subclinically infected herds, respectively. The results demonstrate that<br />
the seroprevalences were higher in the clinically infected herds <strong>and</strong> that most <strong>of</strong> the<br />
pigs became infected with M. hyopneumoniae at a younger age. It can be concluded<br />
that additional factors different from housing <strong>and</strong> management, like differences in<br />
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4 SUMMARY<br />
virulence among M. hyopneumoniae isolates, may determine the infection pattern <strong>of</strong><br />
M. hyopneumoniae <strong>and</strong> the clinical course <strong>of</strong> the infection.<br />
The next chapter (chapter 2.1.2) investigates the virulence <strong>of</strong> M.<br />
hyopneumoniae isolates under experimental conditions. These isolates were obtained<br />
from the herds selected as described in chapter 2.1.1. This study also aimed to link<br />
genetic characteristics, as determined by r<strong>and</strong>omly amplified polymorphic DNA<br />
(RAPD), with the virulence <strong>of</strong> the isolates. Ninety conventional M. hyopneumoniaefree<br />
piglets were inoculated intratracheally with a field isolate, a reference strain <strong>of</strong><br />
high virulence or sterile culture medium. The animals were examined daily for the<br />
presence <strong>of</strong> disease signs <strong>and</strong> a respiratory disease score (RDS) was assessed for<br />
every pig. Twenty-eight days post infection, pigs were euthanized, blood sampled <strong>and</strong><br />
a lung lesion score was given per pig. Lung samples were processed for<br />
histopathology, immun<strong>of</strong>luorescence testing for M. hyopneumoniae <strong>and</strong> isolation <strong>of</strong><br />
M. hyopneumoniae. RAPD analysis was performed on all M. hyopneumoniae isolates.<br />
Significant differences between isolates were found for the RDS, lung lesion<br />
score, histopathology, immun<strong>of</strong>luorescence <strong>and</strong> serology. Based on the results <strong>of</strong> the<br />
different parameters, isolates were divided into three “virulence” groups: low,<br />
moderate <strong>and</strong> high virulence isolates. Typically, a 5000 bp RAPD fragment was<br />
associated with the high <strong>and</strong> moderate virulence isolates whereas it was absent in low<br />
virulence isolates. It was concluded that high variation in virulence exists between M.<br />
hyopneumoniae isolates obtained from different pig herds <strong>and</strong> that further studies are<br />
required to determine whether the 5000 bp b<strong>and</strong> obtained in the RAPD analysis can<br />
be used as a virulence marker.<br />
In the second part <strong>of</strong> this thesis, the susceptibility <strong>of</strong> M. hyopneumoniae to<br />
antimicrobial agents is described. Chapter 2.2.1 reports the in vitro susceptibilities <strong>of</strong><br />
21 M. hyopneumoniae isolates obtained between 2000 <strong>and</strong> 2002. This study was<br />
conducted because information about in vitro susceptibilities <strong>of</strong> M. hyopneumoniae is<br />
scarce. A broth microdilution technique was used to determine the susceptibilities for<br />
12 antimicrobials frequently used in pig herds in Belgium. Acquired resistance to<br />
spectinomycin, oxytetracycline, doxycycline, gentamicin, florfenicol <strong>and</strong> tiamulin<br />
was not observed. One isolate showed acquired resistance to lincomycin, tilmicosin<br />
<strong>and</strong> tylosin. This isolate was susceptible to all other antibiotics tested. The Minimal<br />
Inhibitory Concentration (MIC)-values <strong>of</strong> flumequine were > 16 µg/ml for 5 isolates,<br />
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4 SUMMARY<br />
while the MIC 50 -value was 2 µg/ml. For these 5 isolates, the MIC-values for<br />
enr<strong>of</strong>loxacin were ≥ 0.5 µg/ml, the MIC 50 being 0.06 µg/ml. As far as we know, this<br />
is the first report <strong>of</strong> acquired resistance against macrolides, lincosamides <strong>and</strong><br />
fluoroquinolones in M. hyopneumoniae field isolates.<br />
In the following two chapters (2.2.2 <strong>and</strong> 2.2.3), the mechanisms <strong>of</strong> acquired<br />
resistance <strong>of</strong> M. hyopneumoniae isolates against macrolides, lincosamides <strong>and</strong><br />
streptogramins (MLS) <strong>and</strong> fluoroquinolones were elucidated.<br />
To study the acquired resistance to MLS antibiotics (chapter 2.2.2), the<br />
phenotype <strong>and</strong> the genotype <strong>of</strong> the resistant M. hyopneumoniae field isolate were<br />
compared to 5 susceptible isolates. Additional MICs were determined for other<br />
members <strong>of</strong> the MLS group, eg. erythromycin, clindamycin, azithromycin <strong>and</strong><br />
clarithromycin. The MICs <strong>of</strong> all antibiotics tested were significantly higher for the<br />
resistant isolate. The MICs <strong>of</strong> the 16-membered macrolide tylosin ranged from 8 to 16<br />
µg/ml for the resistant isolate <strong>and</strong> from 0.03 to 0.125 µg/ml for the 5 susceptible<br />
isolates. The MICs for the 15-membered macrolides <strong>and</strong> lincosamides were higher<br />
than 64 µg/ml for the resistant isolate while only 0.06 to 0.5 µg/ml for the susceptible<br />
isolates. M. hyopneumoniae isolates are intrinsically resistant to the 14-membered<br />
macrolides due to a G2057A mutation (E. coli numbering) in their 23S rDNA.<br />
Therefore, high MICs were observed for all isolates, although the MICs for the<br />
resistant isolate were clearly increased. An additional, acquired A2058G point<br />
mutation was found in the 23S rRNA gene <strong>of</strong> the resistant isolate. No differences<br />
linked to resistance were found in the ribosomal proteins L4 <strong>and</strong> L22. The present<br />
study showed that 23S rRNA mutations, resulting in resistance to macrolides <strong>and</strong><br />
lincosamides as described in other Mycoplasma spp., also occur under field conditions<br />
in M. hyopneumoniae.<br />
The mechanism <strong>of</strong> acquired resistance to fluoroquinolones in 5 M.<br />
hyopneumoniae field isolates was studied in chapter 2.2.3. Therefore, the genotypes<br />
<strong>of</strong> the 5 resistant isolates were compared to the genotypes <strong>of</strong> 5 susceptible isolates as<br />
determined in the study described in chapter 2.2.1. Their quinolone resistancedetermining<br />
regions (QRDR) <strong>of</strong> gyrA, gyrB, parC <strong>and</strong> parE were characterized. Parts<br />
<strong>of</strong> the DNA gyrase subunits, gyrA <strong>and</strong> gyrB, <strong>and</strong> the topoisomerase subunits, parC<br />
<strong>and</strong> parE, containing the QRDR, were sequenced. In all 5 resistant isolates, one point<br />
mutation (C → A) in parC was found, resulting in an amino acid change from serine<br />
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4 SUMMARY<br />
to tyrosine at position 80 (E. coli numbering). For 4 <strong>of</strong> these isolates, this was the only<br />
mutation found. These isolates had a MIC <strong>of</strong> enr<strong>of</strong>loxacin <strong>of</strong> 0.5 µg/ml, while for<br />
sensitive isolates the MIC <strong>of</strong> enr<strong>of</strong>loxacin was ≤ 0.06 µg/ml. One resistant isolate (Mh<br />
20) had an extra mutation (C → T) in gyrA resulting in an amino acid change from<br />
alanine to valine at position 83 (E. coli numbering). This mutation was correlated with<br />
an increase in the MIC <strong>of</strong> enr<strong>of</strong>loxacin (> 1 µg/ml). No mutations resulting in an<br />
amino acid change were detected in the QRDR <strong>of</strong> the gyrB <strong>and</strong> parE genes <strong>of</strong> the<br />
selected stains. This is the first description <strong>of</strong> the mechanism <strong>of</strong> resistance against<br />
fluoroquinolones in M. hyopneumoniae.<br />
In the last chapter (chapter 2.2.4), the efficacy <strong>of</strong> in-feed medication with<br />
tylosin (Tylan® Premix; Elanco Animal <strong>Health</strong>) for the treatment <strong>of</strong> enzootic<br />
pneumonia was examined in an experimental M. hyopneumoniae infection model.<br />
Three groups <strong>of</strong> 10 conventional M. hyopneumoniae-free piglets were inoculated<br />
intratracheally with a highly virulent M. hyopneumoniae field isolate (non-treated<br />
control (non-TP) group <strong>and</strong> tylosin premix (TP group)) or with sterile culture medium<br />
(non-infected <strong>and</strong> non-treated control (NC) group). Twelve days after infection when<br />
10% <strong>of</strong> the animals were coughing, tylosin treatment <strong>of</strong> the TP group was initiated<br />
(100 mg/kg <strong>of</strong> feed for 21 days). Animals were daily examined for the presence <strong>of</strong><br />
clinical signs <strong>and</strong> a respiratory disease score (RDS) was given per pig. Thirty-three<br />
days after infection, all pigs were euthanized <strong>and</strong> the lung lesions were quantified.<br />
Lung samples were processed for immun<strong>of</strong>luorescence testing for M. hyopneumoniae.<br />
Average RDS <strong>and</strong> lung lesion scores were significantly higher (P
4 SUMMARY<br />
In conclusion, the most important findings <strong>of</strong> this research are that:<br />
- apart from management <strong>and</strong> housing conditions, the virulence <strong>of</strong> the M.<br />
hyopneumoniae isolate strongly influences the disease-course<br />
- antimicrobial resistance is not a major problem in M. hyopneumoniae isolates<br />
but it does occur in the field <strong>and</strong> careful use <strong>of</strong> antimicrobial agents is<br />
warranted<br />
- the mechanism <strong>of</strong> resistance in M. hyopneumoniae against MLS <strong>and</strong><br />
fluoroquinolones is based on mutations in genes encoding the drug target site<br />
- tylosin (100 ppm) administered in the feed 12 days after experimental<br />
infection is efficient in reducing clinical signs <strong>and</strong> lung lesions<br />
These findings are generally discussed in chapter 3 <strong>and</strong> possibilities for future<br />
research are also indicated.<br />
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- 195 -<br />
5 SAMENVATTING
5 SAMENVATTING<br />
Mycoplasma hyopneumoniae is de primaire oorzaak van enzoötische<br />
pneumonie bij varkens, een chronische ziekte die wereldwijd verantwoordelijk is voor<br />
grote economische verliezen in de intensieve varkenshouderij. In de literatuur wordt<br />
vermeld dat het ziekteverloop en de economische verliezen voornamelijk beïnvloed<br />
worden door de omgevingsfactoren en het bedrijfsmanagement.<br />
In ho<strong>of</strong>dstuk 1.1.2 wordt een overzicht gegeven van de beschikbare<br />
antimicrobiële middelen en hun in vivo en in vitro activiteit tegen M. hyopneumoniae.<br />
Ontwikkeling van antimicrobiële resistentie door Mycoplasma spp. en de gerelateerde<br />
resistentiemechanismen worden samengevat. Voor het bestrijden van enzoötische<br />
pneumonie worden in de praktijk zowel preventieve medicatie als vaccinatie gebruikt.<br />
De voordelen en nadelen van beide strategieën worden bediscussieerd. Tot slot<br />
worden eradicatiestrategieën voor M. hyopneumoniae, die ontwikkeld en toegepast<br />
werden in een aantal l<strong>and</strong>en, besproken.<br />
De voornaamste doelstellingen van deze doctoraatsthesis waren het evalueren<br />
van de virulentie van M. hyopneumoniae veldisolaten en het bestuderen van hun<br />
antimicrobiële gevoeligheid.<br />
In ho<strong>of</strong>dstuk 2.1.1 werden de infectiepatronen van M. hyopneumoniae<br />
onderzocht op 5 bedrijven met een klinisch beeld van enzoötische pneumonie en op 5<br />
subklinisch geïnfecteerde bedrijven. Op de klinisch geïnfecteerde bedrijven waren de<br />
huisvesting en het management goed terwijl deze onvoldoende waren op de<br />
subklinisch geïnfecteerde bedrijven. Op elk bedrijf werd van een aantal<br />
leeftijdscategorieën bloed genomen voor het opsporen van antist<strong>of</strong>fen tegen M.<br />
hyopneumoniae en werden neusswabs verzameld waarin de kiem werd opgezocht<br />
d.m.v. nested PCR (nPCR). Het percentage seropositieve dieren op klinisch<br />
geïnfecteerde bedrijven steeg van 8% bij biggen van 9 weken oud tot 52% bij varkens<br />
van 18 weken. De meeste dieren seroconverteerden tussen 12 en 15 weken leeftijd.<br />
Op de subklinisch geïnfecteerde bedrijven stegen de percentages van 2 naar 24%. De<br />
meeste dieren seroconverteerden tussen 15 en 18 weken. Het percentage nPCR<br />
positieve biggen van 6 weken was 16% op de klinisch geïnfecteerde bedrijven en 0%<br />
op de subklinisch geïnfecteerde bedrijven. Deze resultaten tonen aan dat de<br />
seroprevalenties hoger zijn op klinisch geïnfecteerde bedrijven en dat op deze<br />
bedrijven de meeste biggen geïnfecteerd worden op een jongere leeftijd. Hieruit<br />
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5 SAMENVATTING<br />
kunnen we concluderen dat niet alleen de huisvesting en het management invloed<br />
hebben op het infectiepatroon van M. hyopneumoniae en op het klinisch verloop van<br />
de ziekte. Mogelijks bestaan er ook verschillen in virulentie tussen M. hyopneumoniae<br />
isolaten.<br />
In het volgende ho<strong>of</strong>dstuk (ho<strong>of</strong>dstuk 2.1.2) werd de virulentie van zes M.<br />
hyopneumoniae veldisolaten onderzocht onder experimentele omst<strong>and</strong>igheden. Deze<br />
isolaten werden bekomen op de bedrijven beschreven in ho<strong>of</strong>dstuk 2.1.1. In deze<br />
studie werd ook het verb<strong>and</strong> nagegaan tussen genetische verschillen van de isolaten,<br />
bepaald met behulp van r<strong>and</strong>omly amplified polymorphic DNA (RAPD), en de<br />
virulentie van de isolaten. Negentig conventionele M. hyopneumoniae-vrije biggen<br />
werden intratracheaal geïnoculeerd met een veldisolaat, een hoog-virulente<br />
referentiestam <strong>of</strong> steriel cultuurmedium. Dagelijks werden de klinische symptomen<br />
van de dieren beoordeeld door middel van een “respiratory disease score” (RDS).<br />
Achtentwintig dagen na de infectie werden alle biggen geëuthanaseerd, werd er bloed<br />
genomen en werden de longlesies per varken gescoord. Longstalen werden verwerkt<br />
voor histopathologisch onderzoek, immun<strong>of</strong>luorescentie en isolatie van M.<br />
hyopneumoniae. RAPD analyse werd uitgevoerd op alle M. hyopneumoniae isolaten.<br />
Significante verschillen tussen de isolaten werden waargenomen voor de RDS, de<br />
longlesies, de histopathologie, de immun<strong>of</strong>luorescentie en de serologie. Gebaseerd op<br />
de resultaten van deze parameters werden de isolaten in 3 groepen verdeeld: hoog-,<br />
matig- en laagvirulente isolaten. Opvallend was de aanwezigheid van een 5000 bp<br />
RAPD fragment bij alle hoog- en matig-virulente isolaten. Dit fragment was afwezig<br />
bij laagvirulente isolaten. Uit dit onderzoek kon besloten worden dat er een grote<br />
variatie is in de virulentie van M. hyopneumoniae isolaten die geïsoleerd werden op<br />
de verschillende bedrijven. Bijkomende studies zijn nodig om de 5000 bp b<strong>and</strong> verder<br />
te karakteriseren en om na te gaan <strong>of</strong> deze kan gebruikt worden als virulentiemerker.<br />
In het tweede deel van deze thesis werd ingegaan op de gevoeligheid van M.<br />
hyopneumoniae tegen antimicrobiële agentia. In ho<strong>of</strong>dstuk 2.2.1 werd de gevoeligheid<br />
bepaald van 21 M. hyopneumoniae isolaten, die verzameld werden tussen 2000 en<br />
2002, voor 12 antibiotica die in de Belgische varkenshouderij frequent worden<br />
gebruikt. Hierbij werd gebruik gemaakt van een bouillon microdilutie techniek.<br />
Verworven resistentie tegen spectinomycine, oxytetracycline, doxycycline,<br />
gentamicine, florfenicol en tiamulin werd niet waargenomen. Eén isolaat was resistent<br />
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5 SAMENVATTING<br />
tegen lincomycine, tilmicosine en tylosine, maar gevoelig voor alle <strong>and</strong>ere geteste<br />
antibiotica. De Minimale Inhibitorische Concentratie (MIC)-waarden van flumequine<br />
waren > 16 µg/ml voor 5 isolaten, terwijl de MIC 50 2 µg/ml bedroeg. De MICwaarden<br />
voor enr<strong>of</strong>loxacine waren ≥ 0,5 µg/ml voor deze 5 isolaten, terwijl de MIC 50<br />
0,06 µg/ml bedroeg. Dit is de eerste melding van verworven resistentie tegen<br />
macroliden, lincosamiden en fluoroquinonoles voor M. hyopneumoniae isolaten.<br />
In de volgende twee ho<strong>of</strong>dstukken (2.2.2 en 2.2.3) werden de mechanismen<br />
die verantwoordelijk zijn voor deze verworven resistentie bepaald.<br />
Voor het bestuderen van de verworven resistentie tegen macroliden en<br />
lincosamiden (ho<strong>of</strong>dstuk 2.2.2) werden het fenotype en het genotype van de resistente<br />
M. hyopneumoniae stam vergeleken met 5 gevoelige isolaten. MICs werden bepaald<br />
voor de 14-ring macroliden erythromycine en clarythromycine, het 15-ring macrolide<br />
azithromycine, het 16-ring macrolide tylosine en de lincosamiden lincomycine en<br />
clindamycine. De MIC was voor alle antibiotica significant hoger voor de resistente<br />
stam. De MICs van tylosine varieerden van 8 tot 16 µg/ml voor de resistente stam en<br />
van 0,03 tot 0,125 µg/ml voor de 5 gevoelige isolaten. De MICs van azithromycine en<br />
de lincosamiden waren hoger dan 64 µg/ml voor de resistente stam, maar bedroegen<br />
slechts 0,06 tot 0,5 µg/ml voor de gevoelige isolaten. M. hyopneumoniae isolaten zijn<br />
intrinsiek resistent tegen 14-ring macroliden omwille van een G2057A transitie (E.<br />
coli nummering) in hun 23S rDNA. De MICs van erythromycine waren voor alle<br />
isolaten 8-32 µg/ml. Voor de resistente stam was de MIC nog duidelijk hoger dan<br />
voor de <strong>and</strong>ere isolaten, namelijk >64 µg/ml. Een bijkomende, verworven A2058G<br />
puntmutatie werd gevonden in het 23S rRNA gen van de resistente stam. In de<br />
ribosomale proteïnes L4 en L22 werden geen verschillen teruggevonden die gelinkt<br />
konden worden aan resistentie. Dit onderzoek toont aan dat een mutatie in het 23S<br />
rRNA resulteert in resistentie tegen macroliden en lincosamiden zoals eerder<br />
beschreven voor <strong>and</strong>ere Mycoplasma spp. en dat deze mutatie voorkomt onder<br />
praktijkomst<strong>and</strong>igheden.<br />
Het mechanisme voor verworven resistentie van 5 M. hyopneumoniae isolaten<br />
tegen fluoroquinolones werd verder bestudeerd in ho<strong>of</strong>dstuk 2.2.3. Hiertoe werd het<br />
genotype van de 5 resistente isolaten vergeleken met het genotype van 5 gevoelige<br />
isolaten. De quinolone resistentie bepalende regio’s (“Quinolone resistancedetermining<br />
region: QRDR) van de genen gyrA, gyrB, parC en parE werden<br />
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5 SAMENVATTING<br />
gekarakteriseerd. Hiervoor werden delen van de DNA gyrase subunits, gyrA en gyrB,<br />
en de topoisomerase subunits, parC en parE, die de QRDR bevatten, gesequeneerd. In<br />
de 5 resistente isolaten werd een puntmutatie (C → A) in parC gevonden. De<br />
puntmutatie resulteerde in een aminozuurwijziging van serine naar tyrosine op positie<br />
80 (E. coli nummering). Voor 4 van deze isolaten was dit de enige mutatie die<br />
gevonden werd. De MIC van enr<strong>of</strong>loxacine voor deze isolaten was 0,5 µg/ml terwijl<br />
de MIC van de gevoelige isolaten ≤ 0,06 µg/ml bedroeg. Eén resistente stam met een<br />
MIC van >1 µg/ml voor enr<strong>of</strong>loxacine had een extra mutatie (C → T) in gyrA die<br />
resulteerde in een aminozuurwijziging van alanine naar valine op positie 83 (E. coli<br />
nummering). Mutaties die resulteren in een aminozuurwijziging werden niet<br />
waargenomen in de QRDR van de gyrB en parE genen van de geselecteerde isolaten.<br />
Dit onderzoek beschrijft voor het eerst het mechanisme van fluoroquinoloneresistentie<br />
van M. hyopneumoniae isolaten.<br />
In het laatste ho<strong>of</strong>dstuk (ho<strong>of</strong>dstuk 2.2.4) werd het onderzoek beschreven naar<br />
het effect van voedermedicatie met tylosine (Tylan® Premix; Elanco Animal <strong>Health</strong>)<br />
op het verloop van een experimentele M. hyopneumoniae infectie. Drie groepen van<br />
10 conventionele M. hyopneumoniae-vrije biggen werden intratracheaal geïnoculeerd<br />
met een hoogvirulente M. hyopneumoniae stam die in vitro gevoelig was voor<br />
tylosine (niet-beh<strong>and</strong>elde controle groep (non-TP) en tylosine premix groep (TP)) <strong>of</strong><br />
met steriel cultuurmedium (niet-geïnfecteerde en niet-beh<strong>and</strong>elde controle (NC)<br />
groep). Twaalf dagen na de infectie, op het moment dat 10% van die dieren hoestte,<br />
werd de beh<strong>and</strong>eling met tylosine gestart (100 mg/kg voeder gedurende 21 dagen). De<br />
dieren werden dagelijks gecontroleerd op de aanwezigheid van klinische symptomen<br />
en aan iedere big werd een “respiratory disease score” (RDS) gegeven. Drieëndertig<br />
dagen na inoculatie werden alle biggen geëuthanaseerd en werden de longen<br />
onderzocht op de aanwezigheid van letsels. De gemiddelde RDS en longlesiescores<br />
waren significant hoger in de beide geïnfecteerde groepen vergeleken met de NC<br />
groep. De gemiddelde RDS op 23 tot 33 dagen na infectie was significant hoger<br />
(P
5 SAMENVATTING<br />
De belangrijkste bevindingen van dit doctoraatsonderzoek zijn dat:<br />
- behalve het management en de huisvesting ook de virulentie van de M.<br />
hyopneumoniae stam een sterke invloed heeft op het ziekteverloop van<br />
enzoötische pneumonie<br />
- antibioticumresistentie nog geen groot probleem vormt voor M.<br />
hyopneumoniae isolaten maar wel aanwezig is. Daarom is voorzichtig<br />
omspringen met antimicrobiële middelen noodzakelijk<br />
- het mechanisme voor verworven resistentie van M. hyopneumoniae isolaten<br />
tegen macroliden - lincosamiden en fluoroquinolones berust op mutaties in de<br />
genen die coderen voor de doelwitplaats van deze antimicrobiële middelen<br />
- voerdermedicatie met tylosine (100 ppm) de klinische symptomen en de<br />
longlesies te wijten aan een M. hyopneumoniae infectie, efficiënt reduceert.<br />
Deze bevindingen worden verder bediscussieerd in ho<strong>of</strong>dstuk 3 en<br />
mogelijkheden voor verder onderzoek worden aangegeven.<br />
- 201 -
- 203 -<br />
DANKWOORD
DANKWOORD<br />
Het heeft behoorlijk wat moeite gekost om dit werk te voltooien en op sommige<br />
momenten was een stevige dosis koppigheid hard nodig. Maar nu het zover is, ben ik<br />
bijzonder blij dat ik toch heb volgehouden, want een doctoraat opent wegen waarvan<br />
ik <strong>and</strong>ers maar zou kunnen dromen.<br />
Het is onmogelijk om een doctoraat op je eentje te maken. De vele mensen die hierbij<br />
een groot <strong>of</strong> klein h<strong>and</strong>je hebben bijgedragen wil ik dan ook uitdrukkelijk bedanken.<br />
Allereerst zijn er mijn promotoren, Pr<strong>of</strong>. Haesebrouck en Pr<strong>of</strong>. Maes. Pr<strong>of</strong>.<br />
Haesebrouck, alhoewel ik tijdens de eerste jaren met een klein hartje bij u langs kwam<br />
om door u verbeterde teksten op te halen, heb ik deze verbeteringen en opmerkingen<br />
steeds meer weten te appreciëren. Ze bleken steeds correct en terecht. Soms vraag ik<br />
me af waar u de moed en tijd v<strong>and</strong>aan haalt om telkens weer teksten van verschillende<br />
personen zo nauwgezet te lezen en verbeteren. Daarnaast waardeer ik uw inzicht in en<br />
realistische kijk op het onderzoek, een absolute noodzaak om de experimenten<br />
relevant en uitvoerbaar te houden. Pr<strong>of</strong>. Maes, Dominiek, jij was de persoon bij wie ik<br />
al eens sneller binnen liep met allerh<strong>and</strong>e vragen. Samen met Nathalie en Tim zijn we<br />
de eerste doctor<strong>and</strong>i met u als promotor, en we verdedigen ons doctoraat dan nog in<br />
dezelfde ma<strong>and</strong>. Dominiek, bedankt voor het discussiëren, meedenken, lezen van<br />
teksten,....<br />
Pr<strong>of</strong>. de Kruif, bedankt dat u mij de kans hebt gegeven om aan uw vakgroep te<br />
werken. De vrijheid die u uw assistenten geeft, is zeer stimulerend! Ondanks uw<br />
drukke agenda vond u toch steeds de tijd om interesse te tonen voor mijn onderzoek<br />
en de artikels en ten slotte om dit doctoraat na te lezen. Bedankt ook Pr<strong>of</strong>. em.<br />
Verdonck, Marc, als bezieler van de varkenssektor in deze vakgroep en voor je<br />
interesse in het varkensonderzoek.<br />
Bedankt ook leden van de begeleidings- en examencommissie (Pr<strong>of</strong>. J. Mainil, Pr<strong>of</strong>.<br />
P. Deprez, Pr<strong>of</strong>. P. De Backer, Dr. P. Butaye, Pr<strong>of</strong>. H. Nauwynck en Pr<strong>of</strong>. P.<br />
Wallgren) voor het kritisch lezen van dit doctoraat. Uw opmerkingen droegen zeker<br />
bij tot de kwaliteit ervan.<br />
Dit doctoraat was niet mogelijk zonder de financiële steun van de FOD<br />
Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu. Ik wil in het<br />
bijzonder Dr. X. Van Huffel en Ir. J. Weerts bedanken voor hun vertrouwen in dit<br />
- 205 -
DANKWOORD<br />
onderzoek en voor de stimulerende opmerkingen tijdens de begeleiding van dit<br />
project.<br />
Voor dit onderzoek was er een nauwe samenwerking met het CODA. Deze<br />
samenwerking werd mogelijk gemaakt door Dr. J. Peeters. Mijn eerste collega in het<br />
CODA was Lies Thermote. Lies, het was heel aangenaam om met u samen te werken.<br />
We voelden elkaar goed aan. Het speet me erg toen je vertelde dat je van werk ging<br />
ver<strong>and</strong>eren, maar ik begreep je redenen wel. Gelukkig hebben we contact gehouden.<br />
Het is spijtig dat je er v<strong>and</strong>aag niet kan zijn (het is wat moeilijk om snel even langs te<br />
komen vanuit Vietnam) want jou bijdrage in dit mycoplasma onderzoek is niet gering,<br />
jij bent er immers in geslaagd de eerste M. hyopneumoniae stammen in België te<br />
isoleren. Inge, jij hebt mee dit project uit de grond gestampt en me in de beginfase<br />
mijn weg helpen vinden in de mycoplasma diagnostiek, bedankt. Ook Katrien, en<br />
daarna Saar wil ik bedanken voor hun technische assistentie, vooral voor het<br />
aanmaken van liters NHS20 medium. Tim, jij nam het werk van Lies over en plaatste<br />
het op een sneltrein, alhoewel je eerste ervaringen met het mycoplasma onderzoek<br />
behoorlijk frustrerend waren (5kb...), hoe kan het ook <strong>and</strong>ers als je met M.<br />
hyopneumoniae werkt. Maar je bent er door je dynamiek toch in geslaagd op korte tijd<br />
een mooi doctoraat klaar te krijgen. Ik hoop dat je je weg in het onderzoek kan verder<br />
zetten! Patrick, bedankt om mij wegwijs te maken in het in vitro antibiotica<br />
onderzoek, uw stokpaardje. En nogmaals bedankt voor de tijd die je willen spenderen<br />
hebt in het lezen en bespreken van dit doctoraat.<br />
Een bijzonder woordje van dank wil ik ook richten aan alle varkenshouders die<br />
meegewerkt hebben aan dit onderzoek. Die ons toelieten om stalen te nemen op hun<br />
bedrijven, stalen die noodzakelijk waren voor de isolatie van M. hyopneumoniae, de<br />
basis van dit onderzoek. Ook de familie Malfait ben ik bijzonder dankbaar omdat zij<br />
steeds bereid waren om ons M. hyopneumoniae-vrije biggen te leveren, noodzakelijk<br />
voor de experimentele studies. Daarnaast waren hun slachtvarkens de leveranciers van<br />
bloed, nodig voor het bereiden van medium voor de isolatie van M. hyopneumoniae.<br />
Omdat ik zowel aan de vakgroep Voortplanting, Verloskunde en<br />
Bedrijfsdiergeneeskunde als de vakgroep Pathologie, Bacteriologie en<br />
Pluimveeziekten werkte, had ik veel collega’s. Te veel om hier op te noemen, met het<br />
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DANKWOORD<br />
risico dat ik bepaalde personen zou vergeten. Bedankt collega’s om er te zijn, voor de<br />
steun, voor de hulp, voor het plezier dat we samen hadden, voor het ondergaan van<br />
lastige buien,... En.... badmintonners en lopers, hou vol! Een gezonde geest in een<br />
gezond lichaam zeggen ze toch altijd, hier zit veel waarheid in. Blijf fruit eten....<br />
Een extra woordje van dank aan het ATP van beide vakgroepen, jullie zijn een<br />
onmisbare hulp geweest voor mij.<br />
Marc Coryn, bedankt voor het zeer nauwgezet lezen van dit doctoraat en het<br />
uitfilteren van typ- en spellingsfouten.<br />
Nathalie, met jou heb ik een “samen uit, samen thuis” gevoel. We begonnen samen<br />
aan onze opleiding diergeneeskunde en erna aan ons doctoraat en we verdedigen het<br />
in dezelfde ma<strong>and</strong>. Vanaf nu gaan we <strong>and</strong>ere richtingen uit. Veel succes en plezier,<br />
geniet van je baby! Ook bedankt om gedurende 6 jaar mijn bureaugenootje te zijn.<br />
Tom en Dries, jullie gaan verder met het mycoplasma onderzoek. Veel succes ermee<br />
en laat je niet teveel frustreren.<br />
Sommige aspecten van het werk waren in tegenstelling tot wie ik ben. Zo is uren aan<br />
een computer <strong>of</strong> flow blijven zitten niet echt aan mij besteed. De proefvarkens voeren,<br />
hokken kuisen en eikels voor ze rapen in het bos, om ze toch maar een zo aangenaam<br />
mogelijk leven te geven, ging mij stukken beter af. Maar dan moet je weer afscheid<br />
nemen van dieren, intelligenter dan honden, waarmee je ondertussen een b<strong>and</strong> hebt<br />
opgebouwd. Het werken met Mycoplasma hyopneumoniae vereiste immers<br />
langdurige proeven van minstens 5 weken. Toen ik op het einde van de eerste proef de<br />
varkens moest euthanaseren en hierdoor nogal ongelukkig rondliep was de<br />
boodschap: “de eerste keer voel je je slecht als je je proefdieren moet doden, maar het<br />
went en de 2 de keer gaat het al veel makkelijker”. Bij mij wende dat nooit.<br />
Dirk Lips en Chris De Pauw, ik ben jullie ongelo<strong>of</strong>lijk dankbaar dat jullie me de kans<br />
geven om te werken aan de hogeschool KaHo St.-Lieven in St. Niklaas. Geert<br />
(H<strong>of</strong>lack), jij hebt hier ook een belangrijke rol gespeeld, bedankt! Een reden waarom<br />
ik op mijn t<strong>and</strong>en gebeten heb en dit doctoraat heb afgewerkt, is omdat ik inzag dat<br />
het hebben van een doctoraat een noodzaak zou worden om te kunnen lesgeven aan<br />
een hogeschool. Ik kijk er naar uit om bij jullie te beginnen en hoop dat ik aan jullie<br />
verwachtingen zal kunnen voldoen.<br />
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DANKWOORD<br />
Siegrid, Gwen, Kaat, Ilse, Liesbet, jullie kunnen hier v<strong>and</strong>aag niet zijn omwille van<br />
piepjonge babies (van een babyboom gesproken!). Nu wij nog hé An. “Meiden van<br />
Gent”, ondertussen ben ik nog de enige Gentse, ik hoop dat we er toch nog in slagen<br />
gezellige avondjes en misschien af en toe een weekendje te organiseren. Ik zie jullie<br />
graag.<br />
Lieve, je bent een bijzondere vrouw. Ik heb veel respect voor je!<br />
Cathy en Griet, jullie zijn van onschatbare waarde voor mij. Ik hoop dat jullie dat<br />
beseffen!<br />
Jef, we hebben elkaar dankzij het werk leren kennen en zijn ondertussen vrienden<br />
geworden. Je was een ongelo<strong>of</strong>lijke steun voor mij, ik ben blij dat ik je ken.<br />
Marc en Nicole, we moeten Maarten missen, maar gelukkig hebben we elkaar.<br />
Bedankt voor jullie nimmer aflatende interesse en steun.<br />
Joris, het is dan uiteindelijk toch niet gelukt tussen ons. Een moeilijke beslissing in<br />
een moeilijke periode. Maar het was de moeite waard, een mens groeit in zijn relaties<br />
en in die met jou ben ik veel gegroeid.<br />
En dan mama en papa en Nele en Ward, wat zou ik zijn zonder jullie Jullie merken<br />
het wel hé aan de frequentie dat ik thuis verschijn dat ik niet zonder jullie kan.<br />
Trouwens, thuis is nog steeds Kapellen. Gent is mijn huis.<br />
Jo<br />
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CURRICULUM VITAE
CURRICULUM VITAE<br />
Jo Vicca werd geboren op 31 december 1974 te Tienen. In 1992 beëindigde zij<br />
haar secundaire opleiding aan het Sint-Jozefsinstituut te Tienen in de richting<br />
Wetenschappelijke B. Na 1 jaar de opleiding graduaat chemie te hebben gevolgd aan<br />
de Katholieke Hogeschool Limburg, begon zij in 1993 aan de opleiding tot dierenarts<br />
aan de Faculteit Diergeneeskunde van de Universiteit Gent. Het diploma dierenarts<br />
werd in 1999 behaald met grote onderscheiding. Hierna was zij werkzaam als<br />
wetenschappelijk medewerker in het kader van een onderzoeksproject van de FOD<br />
Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu. Dit project dat<br />
h<strong>and</strong>elde over “de studie van diversiteit van Mycoplasma hyopneumoniae in het kader<br />
van de bestrijding van enzoötische pneumonie” en werd uitgevoerd in samenwerking<br />
met de vakgroepen voortplanting, verloskunde en bedrijfsdiergeneeskunde en<br />
pathologie, bacteriologie en pluimveeziekten van de Faculteit Diergeneeskunde,<br />
Universiteit Gent en het Centrum voor Onderzoek in Diergeneeskunde en<br />
Agrochemie. Het onderzoek uitgevoerd tijdens dit project heeft geleid tot deze<br />
doctoraatsthesis. Jo Vicca is auteur en medeauteur van meerdere wetenschappelijke<br />
publicaties, nam deel aan verschillende wetenschappelijke congressen en gaf<br />
verschillende voordrachten aan dierenartsen en <strong>and</strong>ere onderzoekers.<br />
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PUBLICATIONS
ARTICLES<br />
• Vicca, J., Maes, D.,Thermote, L., Peeters, J.,Haesebrouck, F. & de Kruif, A.<br />
(2002). Patterns <strong>of</strong> Mycoplasma hyopneumoniae infections in Belgian Farrowto-Finish<br />
pig herds with diverging disease course. Journal <strong>of</strong> Veterinary<br />
Medicine, Series B 49, 349-353.<br />
• Maes, D.,Verbeke, W.,Vicca, J.,Verdonck, M. & de Kruif, A. (2003). Benefit to<br />
cost <strong>of</strong> vaccination against Mycoplasma hyopneumoniae in pig herds under<br />
Belgian market conditions from 1996 to 2000. Livestock Production Science<br />
83, 85-93.<br />
• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. &<br />
Haesebrouck, F. (2003). Evaluation <strong>of</strong> virulence <strong>of</strong> Belgian Mycoplasma<br />
hyopneumoniae field isolates. Veterinary Microbiology. 97, 177-190.<br />
• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., de Kruif, A. & Haesebrouck, F.<br />
(2004). In vitro susceptibilities <strong>of</strong> Mycoplasma hyopneumoniae field isolates.<br />
Antimicrobial Agents <strong>and</strong> Chemotherapy 48, 4470-4472.<br />
• Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F. & de Kruif, A.<br />
(2004). Quantification <strong>of</strong> the spread <strong>of</strong> Mycoplasma hyopneumoniae in<br />
nursery pigs using transmission experiments. Preventive Veterinary Medicine<br />
66, 265-275.<br />
• Vicca, J., Maes, D., Jonker, L., de Kruif, A. & Haesebrouck, F. (2005). Efficacy<br />
<strong>of</strong> in-feed medication with tylosin for the treatment <strong>and</strong> control <strong>of</strong><br />
Mycoplasma hyopneumoniae infections. The Veterinary Record 156, 606-610.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., De Baere, T., Verhelst, R.,<br />
Peeters, J., de Kruif, A., Haesebrouck, F. & Vaneechoutte, M. (2005).<br />
Evaluation <strong>of</strong> amplified rDNA restriction analysis (ARDRA) for the<br />
identification <strong>of</strong> Mycoplasma species.BMC Infectious Diseases 5, 46.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Peeters, J., de Kruif, A. &<br />
Haesebrouck, F. (2005). The diversity <strong>of</strong> Mycoplasma hyopneumoniae within<br />
<strong>and</strong> between herds using Pulsed-Field Gel Electrophoresis. Veterinary<br />
Microbiology 10, 29-36.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Imberechts, H., Peeters, J., de Kruif, A.,<br />
Haesebrouck, F. & Maes, D. (2005). A multiplex PCR to identify porcine<br />
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mycoplasmas present in broth cultures. Veterinary Research Communications,<br />
In Press.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Minion, F.C., Peeters, J., de<br />
Kruif, A. & Haesebrouck, F. (2005). Characterization <strong>of</strong> in vivo acquired<br />
resistance <strong>of</strong> Mycoplasma hyopneumoniae to macrolides <strong>and</strong> lincosamides.<br />
Microbial Drug Resistance, 11, 291-295.<br />
• Stakenborg, T., Vicca, J., Verhelst, R., Butaye, P., Maes, D., Naessens, A.,<br />
Claeys, G., De Ganck, C., Haesebrouck, F. & Vaneechoutte, M. (2005).<br />
Evaluation <strong>of</strong> tDNA-PCR for the identification <strong>of</strong> Mollicutes. Journal <strong>of</strong><br />
Clinical Microbiology, 43, 4558-4566.<br />
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POSTERS ON NATIONAL AND INTERNATIONAL CONGRESSES<br />
• Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F. & de Kruif, A.<br />
Patterns <strong>of</strong> Mycoplasma hyopneumoniae infection in Belgian farrow-to-finish<br />
pig herds. 17 th Congress <strong>of</strong> the International Pig Veterinary Society, Ames,<br />
Iowa, USA, 02-05 June 2002.<br />
• Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, <strong>and</strong> F.<br />
Haesebrouck. Evaluation <strong>of</strong> virulence <strong>of</strong> Belgian Mycoplasma hyopneumoniae<br />
Field Isolates. 17 th Congress <strong>of</strong> the International Pig Veterinary Society;<br />
Ames, Iowa, USA, 02-05 June 2002.<br />
• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. &<br />
Haesebrouck, F. Evaluation <strong>of</strong> virulence <strong>of</strong> Belgian Mycoplasma<br />
hyopneumoniae field isolates. 14th international congress <strong>of</strong> the International<br />
Organisation for Mycoplasmology; Vienna, Austria, 07-12 July 2002.<br />
• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A.,<br />
Devriese, L. & Haesebrouck, F. Antibiotic susceptibility <strong>of</strong> Belgian<br />
Mycoplasma hyopneumoniae Field Isolates. 14th international congress <strong>of</strong> the<br />
International Organisation for Mycoplasmology; Vienna, Austria, 07-12 July<br />
2002.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., de Kruif, A. & Haesebrouck, F.<br />
RAPD analysis <strong>of</strong> Belgian Mycoplasma hyopneumoniae strains. 14th<br />
international congress <strong>of</strong> the International Organisation for Mycoplasmology;<br />
Vienna, Austria, 07-12 July 2002.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., de Kruif, A. & Haesebrouck, F.<br />
Development <strong>of</strong> a multiplex PCR to detect Mycoplasmas present in the lungs<br />
<strong>of</strong> pigs. 14th international congress <strong>of</strong> the International Organisation for<br />
Mycoplasmology; Vienna, Austria, 07-12 July 2002.<br />
• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., de Kruif, A. & Haesebrouck, F.<br />
Antimicrobial susceptibilities <strong>of</strong> Mycoplasma hyopneumoniae field isolates.<br />
11 th annual meeting <strong>of</strong> the Flemish society for veterinary epidemiology <strong>and</strong><br />
economics; Torhout, Belgium, 11 December 2003.<br />
• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. &<br />
Haesebrouck, F. In vitro susceptibility <strong>of</strong> Mycoplasma hyopneumoniae field<br />
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isolates. 18 th Congress <strong>of</strong> the International Pig Veterinary Society; Hamburg,<br />
Germany, 27 June-1 July 2004.<br />
• Meyns, T., Maes, D., Vicca, J., Dewulf, J., Haesebrouck, F. & de Kruif, A. Use<br />
<strong>of</strong> transmission experiments to quantify the spread <strong>of</strong> Mycoplasma<br />
hyopneumoniae in nursery pigs. 18 th Congress <strong>of</strong> the International Pig<br />
Veterinary Society, Hamburg, Germany, 27 June-1 July 2004.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., de Kruif, A. & Haesebrouck, F.<br />
Optimisation <strong>of</strong> a Pulsed Field Gel Electrophoresis (PFGE) technique for<br />
Mycoplasma hyopneumoniae. 15th international congress <strong>of</strong> the International<br />
Organisation for Mycoplasmology; Athens, Georgia, USA, 11-16 July 2004.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., de Kruif, A. & Haesebrouck, F.<br />
Characterization <strong>of</strong> a Mycoplasma hyopneumoniae field isolate resistant to<br />
MLS antibiotics. 15th international congress <strong>of</strong> the International Organisation<br />
for Mycoplasmology; Athens, Georgia, USA, 11-16 July 2004.<br />
• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., de Kruif, A. & Haesebrouck, F.<br />
Amplified Fragment Length Polymorphism (AFLP) <strong>of</strong> three porcine<br />
Mycoplasma spp. 15th international congress <strong>of</strong> the International Organisation<br />
for Mycoplasmology; Athens, Georgia, USA, 11-16 July 2004.<br />
• Stakenborg, T., Vicca, J., Verhelst, R., Butaye, P., Maes, D., Naessens, A.,<br />
Clays, G., De Ganck, C., Haesebrouck, F. & Vaneechoutte, M.. Evaluation <strong>of</strong><br />
tDNA-PCR for the identification <strong>of</strong> Mollicutes. Belgian Society <strong>of</strong><br />
Microbiology, Brussels, 3 rd December 2004.<br />
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ORAL PRESENTATIONS<br />
• Vicca, J. Mycoplasma hyopneumoniae infectiepatronen in gesloten<br />
varkensbedrijven, gebruik makende van serologie en nested PCR op<br />
neusswabs. Symposium: Mycoplasma hyopneumoniae, inzichten in<br />
epidemiologie en immunologie, georganiseerd door Groep Geneeskunde van<br />
het Varken en Pfizer Animal <strong>Health</strong>, Ede, Nederl<strong>and</strong>, 20 December 2000.<br />
• Vicca, J. Evaluation <strong>of</strong> virulence <strong>of</strong> Belgian Mycoplasma hyopneumoniae field<br />
isolates. 17 th Congress <strong>of</strong> the International Pig Veterinary Society, Ames,<br />
Iowa, USA, 02-05 June 2002.<br />
• Vicca, J. Onderzoek naar verschillen tussen Belgische Mycoplasma<br />
hyopneumoniae veldisolaten. International Pig Veterinary Society, Belgian<br />
branch studienamiddag, Faculteit Diergeneeskunde, Merelbeke, 22 november<br />
2002.<br />
• Vicca J. La broncho-pneumonie enzootique. Les voor 2de proef,<br />
longa<strong>and</strong>oeningen bij varkens, Faculté de Médécine Vétérinaire, Sart-Tilman,<br />
10 December 2002.<br />
• Vicca, J. Ronde tafel gesprekken Boehringer Ingelheim: infectiepatronen van<br />
Mycoplasma hyopneumoniae. 9x June 2003.<br />
• Vicca, J. Evaluation <strong>of</strong> virulence <strong>of</strong> Belgian Mycoplasma hyopneumoniae field<br />
isolates. Swine <strong>and</strong> wine (vereniging varkensdierenartsen), Roermond,<br />
Nederl<strong>and</strong>, 27 September 2003.<br />
• Vicca, J. Virulentieverschillen en vroege spreiding van M. hyopneumoniae. Post<br />
Academisch Onderwijs in de Diergeneeskunde (PAOD), Houten, Nederl<strong>and</strong>, 1<br />
December 2004.<br />
• Vicca, J. Infecties met Mycoplasma hyopneumoniae. Vakdierenarts varken,<br />
Faculteit Diergeneeskunde, UGent, 8 december 2004.<br />
• Vicca, J. Virulentieverschillen en vroege spreiding van M. hyopneumoniae. Post<br />
Academisch Onderwijs in de Diergeneeskunde (PAOD), Houten, Nederl<strong>and</strong>, 1<br />
december 2004.<br />
• Vicca, J. Virulentieverschillen en vroege spreiding van M. hyopneumoniae. Post<br />
Academisch Onderwijs in de Diergeneeskunde (PAOD), Houten, Nederl<strong>and</strong>, 9<br />
december 2004.<br />
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