view - Department of Reproduction, Obstetrics and Herd Health

FACULTY OF VETERINARY MEDICINE 

Department of Reproduction, Obstetrics and Herd Health 

Department of Pathology, Bacteriology and Poultry Diseases 

Virulence and antimicrobial susceptibility of Mycoplasma 

hyopneumoniae isolates from pigs 

Jo VICCA 

Thesis to obtain the academic degree of Doctor of Veterinary Science (PhD) 

Faculty of Veterinary Medicine, Ghent University 

2005 

Promotoren: Prof. Dr. F. Haesebrouck, Prof. Dr. D. Maes 

Co-promotor: Prof. 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, 

the results did not satisfy and are not included in this PhD thesis. To my opinion, the pigs do deserve a place in this thesis and 

therefore reached the cover. 

BACK COVER: - Me together with my blood donating pigs 

- Scanning electron microscopic photograph of a M. hyopneumoniae colony grown on agar 

- Cryosection of M. hyopneumoniae infected lung tissue labeled with anti-M. hyopneumoniae 

antibodies and showing positive immunofluorescence 

- Section of paraffin embedded M. hyopneumoniae infected lung tissue, stained with hematoxylineosin, 

showing the typical peribronchiolar hyperplasia compressing the involved airway 

PRINTING: Plot-it Merelbeke, 2005 

ISBN: 90-5864-086-8 

EAN: 9789058640864

Wisdom is not a question of learning facts with the mind; it can only be 

acquired through perfection of living. 

N. Sri Ram

CONTENTS 

List of abbreviations 1 

1. INTRODUCTION 3 

1.1 Review of the literature 5 

1.1.1 Pathogenesis of enzootic pneumonia 11 

1.1.2 Control and treatment of enzootic pneumonia in pigs and 

in vitro susceptibility of M. hyopneumoniae 27 

1.2 Aims 59 

2. EXPERIMENTAL STUDIES 63 

2.1 Virulence of Mycoplasma hyopneumoniae isolates 65 

2.1.1 Patterns of Mycoplasma hyopneumoniae infections in Belgian 

farrow-to-finish pig herds with diverging disease course 67 

2.1.2 Evaluation of virulence of Mycoplasma hyopneumoniae field 

isolates 85 

2.2 Susceptibility of Mycoplasma hyopneumoniae to antimicrobial agents 107 

2.2.1 In vitro susceptibilities of Mycoplasma hyopneumoniae field 

isolates 109 

2.2.2 Characterization of in vivo acquired resistance of Mycoplasma 

hyopneumoniae to macrolides and lincosamides 125 

2.2.3 Mechanism of resistance against the fluoroquinoles flumequine 

and enrofloxacin in Mycoplasma hyopneumoniae field isolates 139 

2.2.4 The efficacy of tylosin premix for the treatment and control of 

Mycoplasma hyopneumoniae infections 157 

3 GENERAL DISCUSSION 175 

4 SUMMARY 187 

5 SAMENVATTING 195 

Dankwoord 203 

Curriculum vitae 209 

Publications 213

LIST OF ABBREVIATIONS 

A: Adenine 

ABC: 

ATP-Binding Cassette 

ADG: 

Average Daily weight Gain 

AIAO: 

All-in/All-out 

AMK: 

Amoxycillin 

ANOVA: 

ANalysis Of VAriance 

APC: 

Antigen-Presenting Cell 

APR: 

Apramycin 

ATCC: 

American Type Culture Collection 

ATP: 

Adenosine triphosphate 

BALT: 

Bronchus-Associated Lymphoid Tissue 

bp: 

base pair 

BW: 

Body Weight 

C: Cytosine 

CD: 

Cluster of Differentiation 

CCU: 

Colour Changing Units 

CEF: 

Cefquinome 

CHLO: 

Chloortetracycline 

CIPRO: 

Ciprofloxacin 

CLIN: 

Clindamycin 

COL: 

Colistin 

DANO: 

Danofloxacin 

DNA: 

DeoxyriboNucleic Acid 

DOX: 

Doxycycline 

DWG: 

Daily Weight Gain 

E. coli: Escherichia coli 

ELISA: 

Enzyme-Linked ImmunoSorbent Assay 

ENRO: 

Enrofloxacin 

FCR: 

Feed Conversion Ratio 

FFN: 

Florfenicol 

FLUM: 

Flumequin 

G: Guanine 

GEN: 

Gentamicin 

G-F: 

Growth-Finishing 

HEPA: 

High Efficiency Particulate Air 

IF: 

Immunofluorescence 

Ig: 

Immunoglobulin 

IL: 

Interleukin 

IL-R: 

Interleukin-Receptor 

IM: 

Intramuscular 

IRPCM: 

International Research Programme on Comparative Mycoplasmology 

K+: 

kalium ion 

kDa: 

kilo Dalton 

LIN: 

Lincomycin 

MALP: 

Macrophage Activating LipoProtein 

Mbp: 

Million base pairs 

MCP: 

Monocyte chemoattractant protein 

MEW: 

Medicated Early Weaning 

Mh: 

Mycoplasma hyopneumoniae 

MHC: 

Major Histocompatibility Complex 

M. hyopneumoniae: Mycoplasma hyopneumoniae 

MIC: 

Minimum Inhibitory Concentration 

MIP: 

Macrophage Inflammatory Protein 

MLS: 

Macrolides, Lincosamides and Streptogramins 

MPC: 

Mutant Prevention Concentration 

mRNA: 

messenger RiboNucleic Acid 

- 1 -

N: Nursery 

NCCLS: 

National Committee for Clinical Laboratory Standards 

NK cells: 

Natural Killer cells 

NOR: 

Norfloxacin 

nPCR: 

nested Polymerase Chain Reaction 

OD: 

Optical Density 

OFLO: 

Ofloxacin 

OXY: 

Oxytetracycline 

PBS: 

Phosphate Buffered Saline 

PCR: 

Polymerase Chain Reaction 

PEN: 

Penicillin 

PFGE 

Pulsed Field Gel Electrophoresis 

ppm: 

parts per million 

PRRSV: 

Porcine Reproductive and Respiratory Syndrome Virus 

RAPD: 

Randomly Amplified Polymorphic DNA 

rDNA: 

ribosomal DeoxyriboNucleic Acid 

RDS: 

Respiratory Disease Score 

R n : 

Adjusted reproduction ratio 

RNA: 

RiboNucleic Acid 

rpm: 

revolutions per minute 

RR: 

Repeat Region 

rRNA: 

ribosomal RiboNucleic Acid 

QRDR: 

Quinolone Resistance Determining Region 

30S: 30 Svedberg units 

SD: 

Standard Deviation 

SPIRA: 

Spiramycin 

SPT: 

Spectinomycin 

STR: 

Streptomycin 

SULF: 

Sulfamides 

T: Thymine 

TBE: 

Tris-Borate-EDTA 

TEM: 

Transmission Electron Microscopy 

TET: 

Tetracycline 

Th-cell: 

T helper-cell 

TIA: 

Tiamulin 

TIL: 

Tilmicosin 

TLR: 

Toll-Like Receptor 

TMP: 

Trimethoprim 

TNF: 

Tumor Necrosis Factor 

TP: 

Tylosin-Premix 

tRNA: 

transfer RiboNucleic Acid 

TYL: 

Tylosin 

VAL: 

Valnemulin 

VLA: 

Veterinary Laboratory Agency 

Vlp: 

Variable lipoproteins 

w/v: 

weight/volume 

- 2 -

- 3 - 

1 INTRODUCTION

1.1 REVIEW OF THE LITERATURE 

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Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, a chronic 

respiratory disease occurring worldwide in the pig industry. Over 90% of the Belgian 

pig herds are endemically infected (Maes et al., 1999). The major drawbacks of 

enzootic pneumonia are the economic losses due to decreased daily weight gain, 

increased feed conversion ratio, number of days to reach slaughterweight and 

medication costs. 

Outbreaks of enzootic pneumonia are mainly experienced in the growing and 

finishing units. The incubation period of the disease is 10-16 days under experimental 

conditions, but this period can vary greatly under natural conditions. There is no agerelated 

susceptibility of pigs to infection with M. hyopneumoniae, piglets in the 

farrowing unit as well as sows can become infected after experimental inoculation or 

during a natural outbreak of enzootic pneumonia (Goodwin et al., 1969; Piffer and 

Ross, 1984; Kobisch et al., 1993; Wallgren et al., 1998). 

Macroscopic lesions are mainly found bilaterally in the apical, cardiac, 

intermediate and the anterior parts of the diaphragmatic lobes. They appear at 7-10 

days after experimental infection and reach their maximum extension after 4 weeks. 

The lesions are well demarcated from the normal tissue and consist of purple to gray 

areas of consolidation. After incision of the affected tissue, the consistency is meaty 

and there is a catarrhal exudate in the airways. The bronchial and mediastinal lymph 

nodes are often enlarged. Lesions become deeper red and even more demarcated 

when the disease evolves to the chronic stage. Less exudate is present in the airways. 

Recovering lesions consist of fissures of collapsed alveoli (interlobular scar 

retractions). Macroscopic lesions of a pure M. hyopneumoniae infection are usually 

resolved after 12-14 weeks but M. hyopneumoniae can still be detected (Blanchard et 

al., 1992; Sørensen et al., 1997). Despite the resolving of the lung lesions, the 

organism can be demonstrated in the lungs for at least 185 days using nested PCR on 

bronchial swabs (Fano et al., 2004). 

Because M. hyopneumoniae infections disturb the mucosal clearance, form 

characteristic lesions and modulate the immune system, secondary infections with 

Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, 

Haemophilus parasuis and Streptococcus suis are common (Yagihashi et al., 1984; 

Ciprian et al., 1988; Asai et al., 1996; Sørensen et al., 1997). 

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In the present review, literature dealing with the pathogenesis of enzootic 

pneumonia will be summarized. Thereafter an overview of antimicrobial treatment 

and control of the disease will be presented. 

- 8 -

REFERENCES 

1. Asai, T., Okada, M., Yokomizo, Y., Sato, S. & Mori, Y. (1996). Suppressive effect of 

bronchoalveolar lavage fluid from pigs infected with Mycoplasma hyopneumoniae on 

chemiluminescence of porcine peripheral neutrophils. Veterinary Immunology and 

Immunopathology 51, 325-331. 

2. Blanchard, B., Vena, M.M., Cavalier, A., Le Lannic, J., Gouranton, J. & Kobisch, M. 

(1992). Electron microscopic observation of the respiratory tract of SPF piglets inoculated with 

Mycoplasma hyopneumoniae. Veterinary Microbiology 30, 329-341. 

3. Ciprian, A., Pijoan, C., Cruz, T., Camacho, J., Tortora, J., Colmenares, G., Lopez-Revilla, 

R. & De La Garza, M. (1988). Mycoplasma hyopneumoniae increases the susceptibility of pigs 

to experimental Pasteurella multocida pneumoniae. Canadian Journal of Veterinary Research 

52, 434-438. 

4. Fano, E., Pijoan, C. & Dee, S. (2004). Assessing the duration of Mycoplasma hyopneumoniae 

infection in gilts. Proceedings of the International Pig Veterinary Society, 18 th Congress, June 

27- Juli 1, Hamburg / Germany. 

5. Goodwin, R., Hodgson, R., Wittlestone, P. & Woodhams, R. (1969). Immunity in 

experimentally induced enzootic pneumonia of pigs. Journal of Hygiene (London) 67, 193-208. 

6. Kobisch, M., Blanchard, B. & Le Poitier, M.F. (1993). Mycoplasma hyopneumoniae 

infections in pigs: duration of the disease and resistance to reinfection. Veterinary Research 24, 

67-77. 

7. Maes, D. (1999). Respiratory disease in slaughter pigs: epidemiological aspects and effect of 

vaccination against Mycoplasma hyopneumoniae. PhD Thesis / Ghent University / Belgium. 

8. Piffer, I. & Ross, R. (1984). Effect of age on susceptibility of pigs to Mycoplasma 

hyopneumoniae pneumonia. American Journal of Veterinary Research 45, 478-481. 

9. Sørensen, V., Ahrens, P., Barfod, K., Feenstra, A.A., Feld, N.C., Friis, N.F., Bille-Hansen, 

V., Jensen, N.E. & Pedersen, M.W. (1997). Mycoplasma hyopneumoniae infection in pigs: 

duration of the disease and evaluation of four diagnostic assays. Veterinary Microbiology 54, 

23-34. 

10. Wallgren, O., Bolske, G., Gustafsson, S., Mattsson, S., Fossum, C. (1998). Humoral immune 

responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of 

mycoplasmosis. Veterinary Microbiology 60, 193-205. 

11. Yagihashi, T., Nunoya, T., Mitui, T. & Tajima, M. (1984). Effect of Mycoplasma 

hyopneumoniae on the development of Haemophilus pleuropneumoniae pneumonia in pigs. The 

Japanese Journal of Veterinary Science 46, 705-713. 

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1.1.1 PATHOGENESIS OF ENZOOTIC PNEUMONIA 

- 11 -


Mycoplasmas are the smallest and simplest self-replicating organisms and are 

distinguished phenotypically from other bacteria by their minute size and lack of a 

cell wall. They are characterized by their small genome size (0.58-2.2 Mbp) and a low 

G+C content (23-40 mol% of the genome). During their evolution, Mycoplasma spp. 

have lost all the genes involved in amino acid and fatty acid biosynthesis and thus 

require the full spectrum of the essential amino acids and fatty acids from the host or 

from the artificial culture medium. Also genes involved in cofactor biosynthesis are 

lost, so that to cultivate Mycoplasma spp. in vitro, the medium has to be supplemented 

with essentially all the vitamins (Razin et al., 1998). Competition between 

mycoplasma and host for these biosynthetic precursors may lead to disruption of the 

host cell integrity and alter host cell function (Rottem, 2003). 

ADHESION OF M. HYOPNEUMONIAE TO THE HOST CELLS 

Adhesion of Mollicutes to host cells is a prerequisite for colonization and for 

infection. The loss of adhesion capacity by several passages in vitro results in loss of 

infectivity (Mebus and Underdahl, 1977; Thomas et al., 2003). M. hyopneumoniae 

adheres to the apex of cilia of tracheal, bronchial and bronchiolar epithelial cells but 

does not penetrate into the lung parenchyma nor invade cells (Blanchard et al., 1992; 

Kwon and Chae, 1999). Very few mycoplasmas are present in the small bronchioli 

and alveoli since ciliated epithelium is absent in the lower respiratory tract 

(Christensen et al., 1999). M. hyopneumoniae exclusively adheres to cilia and 

therefore uses fine fibrils (Tajima and Yagihashi, 1982; Blanchard et al., 1992; 

Sarradell et al., 2003) (Figure 1). Using electron microscopy, Tajima and Yagihashi 

(1982) and Tajima et al. (1985) observed the presence of a capsule and found an 

association between the thickness of this polysaccharide capsule and the pathogenicity 

of the organism. 

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Figure 1: Adapted from Tajima and Yagihashi (1982). A TEM photograph of a crosssection 

of the bronchiolar epithelium from a M. hyopneumoniae inoculated pig. 

Numerous mycoplasmas are seen among the cilia and on the tips of microvilli of the 

epithelial cells. The organisms are not in contact with the epithelial surface. Bar: 

1.000 nm. The inset shows a mycoplasma which is elongated and constricted in the 

middle part of the body and appears to be in the process of binary fission. Many radial 

fibrils can be seen projecting outward from the cell surface. Bar: 100 nm. 

Still little is known about the mechanism of adherence of M. hyopneumoniae. 

It is clear that the process is multifactorial and that multiple genes are involved. 

Zhang et al. (1995) identified a ciliary adhesion molecule (P97). Monoclonal 

antibodies against P97 inhibited adherence and stained the fuzzy structures, as 

described by Tajima and Yagihashi (1982), on the surface of the micro-organism. 

Further analysis of the DNA sequences surrounding the P97 structural gene revealed 

an operon composed of two open reading frames encoding the P97 adhesion molecule 

and a 102 kDA protein (P120). The role of P102 is unknown. P97 includes two repeat 

regions, R1 and R2. (Hsu et al., 1997; Hsu and Minion, 1998a). The cilium binding 

site is located in R1 and it is supposed that at least seven AAKPV(E) repeats are 

required for functional binding (Hsu and Minion, 1998b; Minion et al. 2000). 

A glycoprotein of 110 kDa, consisting of one P54 and two P28 subunits is also 

associated with adhesion of M. hyopneumoniae organisms to the cilia (Chen et al., 

1998). 

- 14 -


Adherence is associated with degenerative changes in the ciliated epithelial 

cells: reduction of the ciliary activity, loss of cilia and clumping of cilia. By attaching 

to the surface of host cells, mycoplasmas may interfere with membrane receptors or 

alter transport mechanisms of the host cell. In the case of M. hyopneumoniae, a 

disruption of K+ channels of ciliated bronchial epithelial cells which results in 

ciliostasis has been described (Debey and Ross, 1994). Recent research revealed the 

increase of intracellular calcium in porcine ciliated tracheal cells after adhesion of 

pathogenic M. hyopneumoniae isolates (Park et al., 2002). This increase may serve as 

an intracellular signal to induce loss of cilia. Cell damage may also be caused by the 

mildly toxic by-products of mycoplasma metabolism, such as hydrogen peroxide and 

superoxide radicals. In addition, cytotoxic factors have been associated with some 

Mycoplasma spp., including M. fermentans (Gabridge and Murphy, 1971; Gabridge 

and Schneider, 1975), M. hyorhinis (Darai et al., 1981) M. neurolyticum (Tully, 1981) 

and M. hyopneumoniae (Geary and Walczak, 1985). 

In the host, receptors for mycoplasmas are usually of sialoglycoconjugate 

nature (Razin and Jacobs, 1992; Zhang et al., 1994). The restricted attachment of M. 

hyopneumoniae to cilia can partially be explained by the presence of these 

sialoglycoconjugate-type receptors on the apical microvillar border and the cilia, and 

the absence on the secretory cells and mucus (Loveless and Feizi, 1989). 

MICROSCOPIC LESIONS ASSOCIATED WITH M. HYOPNEUMONIAE INFECTION 

Histological lesions in the acute stage of the disease include accumulation of 

neutrophils and macrophages in and around airways (Livingston et al., 1972). As the 

disease progresses (7-28 days post infection), the changes consist of infiltrates of 

mononuclear cells (lymphocytes and macrophages) around bronchi, bronchioles and 

small blood vessels, accumulation of inflammatory mononuclear cells forming 

lymphoid follicles around the airways and prominent lymphoid hyperplasia of the 

lymphoid tissue associated with intrapulmonary airways, known as bronchusassociated 

lymphoid tissue (BALT) (Huang et al., 1990) (Figure 2). In alveolar 

lumina and septa, lymphocytes, plasma cells and neutrophils accumulate (Rodríguez 

et al., 2004). This accumulation aggravates obliteration of the lumen of bronchioles 

and atelectasis of surrounding alveoli. 

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Figure 2: Infection with M. hyopneumoniae is characterized by infiltration of 

mononuclear cells around bronchi, bronchioles and small blood vessels forming 

lymphoid follicles. This accumulation aggravates obliteration of the lumen of bronchi 

and bronchioles (arrow) and small blood vessels. Hematoxilin-Eosin X 400. 

Four mechanisms may contribute to the obliteration of airways: (1) 

accumulation of mucus and inflammatory exudates due to loss of mucociliary 

function, (2) increased activity of mucus-secreting cells and altered glycoprotein 

production in goblet cells, (3) broncho-constriction by chemical mediators released by 

alveolar macrophages and inflammatory cells and (4) pressure from the aggregates of 

lymphoid tissue (Sarradell et al., 2003). 

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INTERACTION OF M. HYOPNEUMONIAE WITH THE IMMUNE SYSTEM 

The complex network of interactions between mycoplasmas and the host 

immune system involves mycoplasma-induced non-specific and specific immune 

reactions. Non-specific mechanisms include (1) increase of the cytotoxicity of 

macrophages, natural killer cells and T-cells, (2) enhancement of the expression of 

cell receptors and activating the complement cascade and (3) polyclonal activation of 

B- and T-lymphocytes. Specific protective defense mechanisms include (1) 

stimulation of cell-mediated immunity, (2) opsonisation and phagocytosis of 

organisms and (3) the production of systemic as well as local anti-mycoplasmal 

antibodies of different classes and subclasses. These immune reactions are important 

in host defense but have also been shown to play a role in the development of lesions 

and the exacerbation of mycoplasma induced disease (Razin et al., 1998). 

After colonizing the respiratory tract, M. hyopneumoniae stimulates 

macrophages to secrete cytokines (Asai et al., 1993, Rodríguez et al., 2004). Proinflammatory 

cytokines (IL-1, IL-6 and TNF-α) exert a non-specific mitogenic 

activity of M. hyopneumoniae on lymphocytes. 

Lymphocyte recruitment and activation are important in the pathogenesis of 

mycoplasma respiratory disease. T-cells are an important component of these 

responses. In M. hyopneumoniae infected animals, CD4+ Th-cells represent the 

predominant subset responsible of the observed BALT hyperplasia (Saradell et al., 

2003). CD4+ Th-cells are capable of activating macrophages, which results in 

efficient intracellular killing of the mycoplasmas (Caruso and Ross, 1990; Messier et 

al., 1990; Asai et al., 1994). Using in situ hybridization, Kwon and Chae (1999) were 

able to demonstrate the presence of M. hyopneumoniae DNA in interstitial and 

alveolar macrophages. In mice, CD4+ Th-cells play a role in immune mediated 

pathogenesis of mycoplasmal disease since CD4+ Th-cell depleted mice develop 

milder pulmonary lesions than immunocompetent mice (Jones et al., 2002). Both 

Th1- and Th2-type cytokine responses develop in mycoplasma respiratory disease and 

these responses are associated with disease severity. Infection results in a shift from 

the resident Th2 population to a mixed Th1/Th2 response. CD8+ T- 

- 17 -


cells are also observed in M. hyopneumoniae infected lungs (Sarradell et al., 2003), 

but less numerous compared to the CD4+ subpopulation. These cells were shown to 

dampen mycoplasma-associated proinflammatory responses during in vivo 

experimental studies in mice (Jones et al., 2002). The recruitment of B-lymphocytes 

results in locally secreted IgA en IgG (Messier et al., 1990). IgA prevents the 

adhesion of mycoplasmas to the ciliated epithelium, and IgG participates in 

opsonization and phagocytosis by alveolar macrophages (Sheldrake, 1990; Sheldrake 

et al., 1993; Walker et al., 1996). Local humoral immune response can be protective 

before exposure to mycoplasmas (Okada et al., 2000; Fagan et al., 2001) or can 

prevent the spread of the organism to other tissues. Locally secreted antibodies appear 

to play a limited role in recovery from mycoplasma infection since mycoplasmas can 

survive despite vigorous local antibody responses in the host (Simecka et al., 1993; 

Djordjevic et al., 1997). For several respiratory mycoplasmal diseases, locally 

produced antibodies are more important than circulating antibodies (Hodge and 

Simecka, 2002). 

Recently, research aimed to further clarify the trigger for inflammatory cell 

influx after M. hyopneumoniae infection by studying the role of pro-inflammatory 

cytokines, which are believed to play a prominent role in the disease symptoms of 

enzootic pneumonia. Lipoproteins are probably the principle components of intact 

mycoplasma organisms that activate macrophages and they may play an important 

role in the inflammatory process (Chambaud et al., 1999). After interaction with 

ciliated epithelial cells, M. hyopneumoniae causes an increase of the proinflammatory 

cytokines IL-1, IL-6 and TNF-α (Asai et al., 1993; 1994; 

Thanawongnuwech et al., 2001; Rodríguez et al., 2004). The main biological actions 

of the pro-inflammatory cytokines are summarized in Table 1. 

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Table 1: Biological actions of IL-1, IL-6 and TNF-α (adapted from Henderson et al., 

1996). 

Biological action 

Action displayed by: 

IL-1 IL-6 TNF-α 

Actions involved in lesion development after infection with M. hyopneumoniae 

Activation of T- and B-lymphocytes + + + 

Stimulation of immunoglobulin synthesis - + - 

Upregulation of cytocidal activity of macrophages and + - + 

NK cells 

Increased expression of MHC antigen on APC + - + 

Stimulation of cyclooxygenase II induction + - + 

Activation of endothelial cells + - + 

Induction of endothelial adhesion molecules + - + 

Induction of IL-1, TNF-α and IL-8 + - + 

Induction of IL-6 + - + 

Induction of IL-2 and IL-2R - + - 

Endogenous pyrogen + + + 

Additional actions with no direct impact on lesion development after infection 

with M. hyopneumoniae 

Induction of acute-phase proteins + + + 

Stimulation of fibroblast proliferation + + + 

Stimulation of hematopoiesis + + + 

Stimulation of cartilage breakdown + - + 

Stimulation of murine bone breakdown + + + 

Induction of septic-shock like syndrome + - + 

Induction of hyperalgesia + - + 

A macrophage activating lipoprotein 2 kDa (MALP-2), characterized in 

Mycoplasma fermentans (Mühlradt et al., 1997), was found to be a potent inducer in 

vitro and in vivo of the chemokines macrophage inflammatory protein 1α (MIP-1α), 

monocyte chemoattractant protein 1 (MCP-1) and MIP-2 (Deiters and Mühlradt, 

1999, Lührmann et al., 2002). This diacylated lipoprotein, integrated in the membrane 

of mycoplasmas, is recognized by Toll-like receptors (TLR)2 / TLR6. This 

recognition leads to a cascade reaction resulting in apoptotic cell death, complement 

activation or cytokine production (Akira and Hemmi, 2003; Into et al., 2004). TLR 

families expressed in the antigen-presenting cells (APCs) are pattern recognition 

molecules that recognize foreign antigens during innate immune responses of the host 

- 19 -


animals. MALP-2 like structures have been found in other mycoplasmas as well, 

including M. arthritidis (Cole, 1991), M. hyorhinis (Mühlrath et al., 1998) and M. 

salivarum (Shibata et al., 2000; Okusawa et al., 2004). Several lipoproteins in M. 

hyopneumoniae have been described: p65, p50 and p44 (Wise and Kim, 1987). The 

recently sequenced genome of M. hyopneumoniae revealed 53 open reading frames 

with prokaryotic lipoprotein lipid attachment sites, but only 3 of them have a 

functional assignment (Minion et al., 2004). P46 and P65 have already been studied 

and found to be strong species-specific immunogenic proteins (Futo et al., 1995; 

Schmidt et al., 2004). P65 has lipolytic properties (Schmidt et al., 2004). The 

presence of TLR2 and TLR6 on porcine alveolar macrophages was demonstrated by 

Muneta et al. (2003). The same authors also demonstrated that blocking TLR2 and 

TLR6 by monoclonal antibodies resulted in a decrease of TNF-α production by 

alveolar macrophages in vitro. The blocking did not completely inhibit TNF-α 

production, suggesting that other receptors may intervene in cytokine production 

(Muneta et al., 2003). 

Several studied mycoplasmas have the tendency to persist for long periods at 

the site of infection despite of the presence of a strong immune response (Adegboye, 

1978). M. hyopneumoniae has been demonstrated in pig lungs until 185 days after 

infection (Fano et al., 2004). The ability to polyclonally activate cells of the immune 

system may be one possible mechanism to modulate immunity and avoid clearance 

(Simecka, 2005). Another approach that mycoplasma may use to avoid clearance is 

through the variation of cell surface antigens. The best known mechanism is 

phenotypic plasticity by antigenic variation (Rottem, 2003). The number of genes 

involved in diversifying the antigenic nature of their cell is relatively high compared 

to other bacteria (Razin et al., 1998). The system used for this surface variation has 

been described in M. hyorhinis, a gene family called vlp genes, in M. gallisepticum 

(pMGA genes), M. bovis (vsp genes) and some human mycoplasmas (Raizin et al., 

1998). Mostly lipoproteins are involved. Genes for lipoproteins contain tandem 

repeats that undergo slipped strand mispairing, resulting in either phase switching or 

phase variation. A similar system has not (yet) been described for M. hyopneumoniae. 

Compared to other Mycoplasmas spp., M. hyopneumoniae has few significant tandem 

repeat sequences, with one exception, the gene for the cilium adhesin P97, as 

described earlier (Djordjevic et al., 2004; Minion et al., 2004). 

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REFERENCES 

1. Adegboye, D.S. (1978). A review of Mycoplasma-induced immunosuppression. British Veterinary 

Journal 134, 556-560. 

2. Akira, S. & Hemmi, H. (2003). Recognition of pathogen-associated molecular patterns by TLR 

family. Immunology Letters 85, 85-95. 

3. Asai, T., Okada, M., Ono, M., Irisawa, T., Mori, Y., Yokomizo, Y. & Sato, S. (1993). 

Increased levels of tumor necrosis factor and interleukin-1 in bronchoalveolar lavage fluids from 

pigs infected with Mycoplasma hyopneumoniae. Veterinary Immunology and Immunopathology 

38, 253-260. 

4. Asai, T., Okada, M., Ono, M., Mori, Y., Yokomizo, Y. & Sato, S. (1994). Detection of 

interleukin-6 and prostaglandin E2 in bronchoalveolar lavage fluids of pigs experimentally 

infected with Mycoplasma hyopneumoniae. Veterinary Immunology and Immunopathology 44, 

97-102. 

5. Blanchard, B., Vena, M.M., Cavalier, A., Le Lannic, J., Gouranton, J. & Kobisch, M. (1992). 

Electron microscopic observation of the respiratory tract of SPF piglets inoculated with 

Mycoplasma hyopneumoniae. Veterinary Microbiology 30, 329-341. 

6. Caruso, J.P. & Ross, R.F. (1990). Effects of Mycoplasma hyopneumoniae and Actinobacillus 

(Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine. 

American Journal of Veterinary Research 51, 227-231. 

7. Chambaud, I., Wróblewski, H. & Blanchard, A. (1999). Interactions between mycoplasma 

lipoproteins and the host immune system. Trends in Microbiology 7, 493-499. 

8. Chen, J.R., Lin, J.H., Weng, C.N. & Lai, S.S. (1998). Identification of a novel adhesion-like 

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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF 

ENZOOTIC PNEUMONIA IN PIGS 

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1.1.2 ANTIMICROBIAL TREATMENT AND CONTROL OF ENZOOTIC PNEUMONIA IN PIGS 

Enzootic pneumonia is responsible for large economic losses in the pig 

industry worldwide. This makes prevention or treatment of the disease inevitable to 

limit the losses. Control of mycoplasmal infections can be accomplished by 

optimizing of management and housing, by vaccination and by preventive medication. 

Improvement of the housing and management conditions (ventilation, stocking 

density, purchase policy and hygiene) are primordial in the control of enzootic 

pneumonia and should be the first to be accomplished. In a well managed herd still 

suffering from M. hyopneumoniae infections, vaccination is a further important tool to 

reduce economic losses. Vaccination against M. hyopneumoniae in pigs is 

successfully practiced in most countries with an intensive pig production (Maes et al., 

1998; 1999; 2003; Haesebrouck et al., 2004). Local, mucosal, humoral and cellular 

immune responses are induced, resulting in a reduction of lung lesions (Thacker et al., 

2000). Economical benefit is achieved by an increase in daily weight gain (Jensen et 

al., 2002), a decrease of the feed conversion ratio and a reduction in medication costs 

(Maes et al., 2003). 

Nevertheless, the use of antimicrobials, either in a preventive or curative way 

remains necessary in many pig herds, due to a non-vaccination policy, vaccination 

failure or purchase of piglets with unknown vaccination status or from several 

suppliers. The control of the disease by medication can be approached by strategic 

administration of antimicrobials. This paper gives an overview of antimicrobials 

active against M. hyopneumoniae and summarizes in vitro and in vivo studies 

conducted to evaluate that activity. 

ANTIMICROBIAL AGENTS USED FOR THE TREATMENT OF M. 

HYOPNEUMONIAE INFECTIONS 

Potentially active antimicrobials against M. hyopneumoniae include 

tetracyclines, macrolides and lincosamides, pleuromutilins, fluoroquinolones, 

florfenicol, aminoglycosides and aminocyclitols. To control and treat respiratory 

disease in swine, tetracyclines and macrolides are most frequently used (Chauvin et 

al., 2002; Timmerman et al., 2005). Only fluoroquinolones and aminoglycosides have 

mycoplasmacidal effect (Hannan et al., 1989) which is an important characteristic 

when eradication of enzootic pneumonia is intended. 

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In Table 1, recommended systemic or oral dosages of antimicrobials used to 

treat Mycoplasma hyopneumoniae infections are summarized. 

Table 1: Recommended systemic / oral dosages of antimicrobials used to treat 

Mycoplasma hyopneumoniae infections in pigs. 

Type of 

antimicrobial 

Route of 

administration 

Dose (mg/kg 

bodyweight) 

Interval (h) 

Oxytetracycline IM 

Oral 

10 - 20 

20-50 

12 - 24 

- 

Doxycyline hyclate Oral 10 

Lincomycin 

hydrochloride 

IM 

Oral 

10 

5-10 

24 

- 

Tilmicosin Oral 8-20 - 

Tylosin 

IM 

Oral 

5-20 

30-60 

24 

- 

Tulathromycin IM 2.5 - 

Tiamulin Oral 8.8 - 

Valnemulin Oral 3-4 - 

Florfenicol IM 15 48 

Enrofloxacin IM 2.5-5 24 

Marbofloxacin IM 2 24 

Flumequin Oral 15 - 

Gentamicin IM 3-5 24 

Spectinomycin IM 20 12 

1. Tetracyclines 

Tetracyclines (including tetracycline, chlortetracycline, doxycycline, and 

oxytetracycline) are amphoteric molecules that are poorly soluble in water at pH 7. 

Tetracyclines are bacteriostatic antibiotics that bind irreversibly to receptors of the 

30S bacterial ribosome, where they interfere with the association of aminoacyl-tRNA 

to the acceptor site on the mRNA ribosome complex resulting in inhibition of 

bacterial protein synthesis (Prescott, 2000b; Chopra and Roberts, 2001). They are 

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broad-spectrum antimicrobials active against Gram-positive bacteria, including 

mycoplasmas, and Gram-negative bacteria, including chlamydiae and rickettsias, and 

some protozoa (Babesia, Theileria). In the host, their bacteriostatic activity is optimal 

in an acid environment. Oral bioavailability is lowered when administered in feed, 

except for doxycycline. Bivalent cations (calcium, magnesium, iron) decrease the 

absorption and activity by chelating tetracycline (Luthman and Jacobsson, 1983). 

Also, when administered via the drinking water, the bioavailability can be influenced 

by the water quality (degree of acidity) and bivalent cations in the material (iron) of 

the water pipes (Prescott, 2000b). Tetracyclines are lipophilic and therefore diffuse 

easily through biological barriers and cell membranes. Generally speaking, 

tetracyclines are the drugs of first choice for the treatment of respiratory Mycoplasma 

infections in pigs. 

2. Macrolides and lincosamides 

Macrolides and lincosamides are structurally distinct antibiotics but share 

many common properties such as a similar mode of action and an overlapping binding 

site on the 50S ribosome (Weisblum, 1998; Vester and Douthwaite, 2001). They are 

basic compounds, have a high lipid solubility and have a good absorption from the 

intestine and tissue distribution. After uptake, macrolides and lincosamides 

accumulate mainly in phagocytes, with 51 - 85 % localized in lysozomes (Scorneaux 

and Shryock, 1998). Like tetracyclines, macrolides and lincosamides inhibit protein 

synthesis at the ribosome level and are bacteriostatic. By binding to the 50S subunit of 

the ribosome, they block the growth of the nascent peptide chain, probably causing 

premature dissociation of the peptidyl-tRNA from the ribosome (Vester and 

Douthwaite, 2001). Macrolides and lincosamides have been mapped by chemical 

footprinting to the central loop in domain V of 23S rRNA, which is associated with 

the peptidyl transferase activity (Cundliffe, 1990). 

Macrolides are divided into three subgroups according to the size of their 

lactone ring. Erythromycin and oleandomycin belong to the 14-membered ring 

macrolides, while josamycin, kitasamycin, tylosin, tilmicosin and spiramycin belong 

to the 16-membered ring subgroup. Azithromycin and tulathromycin are members of 

the 15 membered ring macrolides. Macrolides are active against Gram-positive 

bacteria (Rhodococcus equi, Bacillus spp., Corynebacterium spp., Erysipelothrix 

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rhusiopathiae, Listeria spp., Staphylococcus spp., Streptococcus spp., Mycoplasma 

spp.), selected Gram-negative bacteria (Pasteurella, Mannheimia, Leptospira, 

Campylobacter, Actinobacillus) and anaerobes (Actinomyces spp., Clostridium spp., 

Bacteroides spp., Prevotella spp., …). 

Lincosamides (lincomycin and clindamycin) are active against many Grampositive 

bacteria and anaerobes but they are considerably less effective against Gramnegative 

bacteria and Mycoplasma spp. compared to macrolides (Prescot, 2000c). 

Absorption of lincomycin and clindamycin is very good after oral administration in 

monogastric animals. Absorption is slower when administered together with food. 

Lincosamides diffuse well through biological barriers and can obtain high tissue 

concentrations. 

3. Pleuromutilins 

Pleuromutilins (tiamulin and valnemulin) share many common properties such 

as a similar mode of action and an overlapping binding site on the 50S ribosome 

(Weisblum, 1998; Vester and Douthwaite, 2001) with macrolides and lincosamides. 

They are bacteriostatic, have a comparable antimicrobial spectrum as macrolides, but 

have remarkable activities against anaerobes and mycoplasmas. Pleuromutilins are 

exclusively used in veterinary medicine, mainly in swine. Valnemulin is 30 times 

more active than tiamulin, especially against Mycoplasma spp. (Aitken et al., 1999). 

Pleuromutilins are almost completely absorbed after oral administration in 

monogastrates (Prescott, 2000c). The bioavailability of both compounds reaches over 

90% and both achieve high drug concentrations in the lung (Burch, 2001). 

4. Fluoroquinolones 

Fluoroquinolones are widely used broad-spectrum antibiotics in human and 

veterinary medicine. Nalidixic acid, the original non-fluorinated molecule of the 

quinolone class, is inactive against Gram-positive bacteria and Mycoplasma spp. 

(Roberts, 1992). These micro-organisms are however susceptible to the derived 

fluoroquinolone classes. Compared to flumequine, the fluoroquinolones enrofloxacin, 

danofloxacin, ciprofloxacin and norfloxacin show an enhanced activity against all 

- 32 -


mycoplasmas studied (Hannan et al., 1997a) and are mycoplasmacidal when they are 

administered in an increased dose (Arai et al., 1992). 

The intracellular targets of fluoroquinolones in bacteria are considered the 

type II topoisomerases, DNA gyrase and topoisomerase IV. Both enzymes are 

essential for DNA replication. DNA gyrase is composed of two GyrA and two GyrB 

subunits (A 2 B 2 ), encoded by the gyrA and gyrB genes, respectively. This tetrameric 

enzyme catalyzes ATP-dependent negative supercoiling of DNA. Topoisomerase IV, 

a C 2 E 2 tetramer encoded by the parC and parE genes, separates interlocked DNA 

strands to allow segregation of daughter chromosomes into daughter cells. ParC is 

similar in structure to GyrA while ParE is homologous to GyrB (Hooper, 2002). 

In monogastric animals, orally administered fluoroquinolones are absorbed quickly 

and completely (80-100%) (Inui et al., 1998). Maximum plasma concentrations are 

reached one hour after intake. Fluoroquinolones are strongly lipophilic and therefore a 

high tissue distribution is reached with good penetration through biological barriers. 

5. Florfenicol 

Florfenicol is exclusively used in veterinary medicine. The compound is 

analogous to chloramphenicol but does not cause irreversible depression of the bone 

marrow and can therefore be used in food producing animals. Florfenicol is a strong 

inhibitor of the microbial protein synthesis through irreversible binding with the 50S 

subunit of the ribosomes, abolishing the activity of peptidyl transferase. The antibiotic 

is bacteriostatic and active against most Gram-positive (including Mycoplasma spp.) 

and Gram-negative bacteria (including rickettsia and chlamydia) and anaerobes. 

Pharmacokinetic data on florfenicol metabolism in the pig are scarce, but the 

properties are generally similar to chloramphenicol. This lipophilic drug has a wide 

tissue distribution. Florfenicol is absorbed quickly and completely after oral 

administration, rapidly distributed and eliminated slowly. Feed intake did not 

significantly change the pharmacokinetics of florfenicol after oral administration in 

pigs (Liu et al., 2003). 

- 33 -


6. Aminoglycosides and aminocyclitols 

Aminoglycosides (streptomycin, neomycin, kanamycin, gentamicin, 

tobramycin, apramycin and amikacin) and aminocyclitols (spectinomycin) are a 

widely used group of broad-spectrum antimicrobials which are bactericidal and target 

the bacterial ribosome. They inhibit bacterial protein synthesis by binding on one 

(streptomycin) or different sites in the 16S rRNA of the 30S subunit and to some 

ribosomal proteins, decreasing the fidelity of translation (Kotra et al., 2000). Other 

effects of aminoglycosides include interference with the cellular electron transport 

system, induction of RNA breakdown, inhibition of translation, effects on DNA 

metabolism and damage to cell membranes (Prescott, 2000a). They are active against 

several Gram-positive (including Mycoplasma spp. and Mycobacterium spp.) and 

many Gram-negative bacteria. Their action against Streptococcus spp. is limited. The 

activity decreases in an anaerobic, acid or purulent environment since penetration 

through the cell membrane is in part an active oxydative transport. 

Aminoglycosides are administered orally or parenterally. Resorption is limited 

in the gastro-intestinal tract and they bind poorly to plasma proteins (less than 25%). 

Distribution after parenteral administration is limited to the extracellular fluid since 

aminoglycosides in general are too polar to cross mammalian cell membranes by 

diffusion. Poor diffusibility can be attributed to their low degree of lipid solubility 

(Giroux et al., 1995). Parenterally administered aminoglycosides easily penetrate in 

lung parenchyma and bronchial secretions according to the plasma-tissue 

concentration ratio (Saux et al., 1986; Goldstein et al., 2002). 

ROUTE OF ANTIMICROBIAL ADMINISTRATION 

In modern pig herds, animals are mostly treated as a group and not as single 

individuals. Consequently, treatment occurs predominantly through in-feed or inwater 

medication because this is easier to apply and less stressful compared to 

parenteral medication. Usually, only pigs suffering from acute disease receive 

parenteral injections on the first 2-3 days of disease, and are usually further treated 

per os. Parenteral injection often results in the best response, particularly in the case 

of acute respiratory tract disease (Henry and Apley, 1999). 

- 34 -


Achieving sufficiently high levels of the administered drug in the lung tissue is 

difficult when using oral medication. Even though in-feed medication is the most 

common method of administering antimicrobials, there are several disadvantages. 

This approach is rather wasteful since diseased but also healthy pigs receive 

medication. This can be justified by the knowledge that penmates are at-risk to 

become infected as well, especially in herds with a high stocking density. Sick pigs 

often have a suppressed appetite and therefore do not eat sufficient amounts of the 

drug to reach therapeutic levels in their body. Adaptation of the antimicrobial dose in 

the feed or water may overcome this problem. A close follow-up of the feed intake is 

required to achieve appropriate therapeutic levels and to avoid toxic effects due to 

overdosing. Some antimicrobial agents are poorly absorbed or are metabolized by the 

low pH in the stomach and may therefore not achieve therapeutic levels. The choice 

of a correct antimicrobial is indispensable. In-feed medication is especially suited for 

endemic or chronic diseases, such as enzootic pneumonia, since a quick 

administration of the medicated feed is not as important as for acute infections with 

eg. Actinobacillus pleuropneumoniae (Henry and Apley, 1999; Friendship, 2000). 

Pulse-medication is a variant of in-feed or in-water medication (Burch, 1990). A short 

period during which a therapeutic dose of antimicrobials is included in the feed is 

alternated with a longer period without medication. During the non-medication period 

pigs are able to develop an active immune response. 

In-water medication which can be administered more quickly is recommended 

for acute diseases, such as Actinobacillus pleuropneumoniae infections. Sick pigs 

often continue to drink, even when they refuse food. A major disadvantage is the 

spilling that often occurs by playing with the drinking nipples out of boredom. Some 

antimicrobials are unstable in water, poorly soluble due to the water pH or react with 

the water pipe material (e.g. tetracyclines with iron). Water intake of the piglets may 

greatly vary with the season; appropriate follow-up of the water intake is a 

prerequisite for achieving correct therapeutic doses (Henry and Apley, 1999). 

M. hyopneumoniae infections can be managed metaphylaxially or 

prophylaxially. Metaphylaxis means the use of medication with therapeutic levels of 

drugs when some animals are clinically diseased while others are subclinically 

diseased or at high risk. Prophylaxis is the use of medication with therapeutic levels 

of drugs during high-risk periods for disease. A high-risk period in Western Europe 

- 35 -


for achieving M. hyopneumoniae infection is usually at 10 weeks of age, after transfer 

of animals to the finishing houses (Léon et al., 2001). 

THE EFFICACY OF SEVERAL ANTIMICROBIALS AGAINST M. HYOPNEUMONIAE 

INFECTIONS UNDER EXPERIMENTAL AND / OR FIELD CONDITIONS. 

Several studies have been conducted to assess the efficacy of various 

antimicrobials used for the prevention and the treatment of M. hyopneumoniae 

infections. Comparison of these studies is complicated due to considerable variations 

in study design. An overview of the conclusions drawn from studies published in 

peer-reviewed journals is given in Table 2 (experimental studies) and Table 3 (field 

studies). 

From these tables it can be concluded that for most antimicrobials tested, 

economic parameters (daily weight gain and feed conversion ratio) were improved 

and lung lesions as well as clinical signs were decreased in treated animals. 

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Table 2: The effect of different antimicrobial regimens for treatment of experimental 

infections with Mycoplasma hyopneumoniae. 

Antimicrobial drug, dosage, 

scheme 

6-chloro analogue of Norfloxacin 

400 or 200 ppm 

Norfloxacin 100 ppm 

feed medication during 3 weeks 

starting 1 month after infection 

Tiamulin 100 or 200 ppm 

feed medication during 10 days 

starting 2 days before infection 

Tiamulin 50 mg/kg 


starting 1 month after infection 

Tiamulin 240 ppm 


starting 10 days after infection 

Tiamulin 60, 120, 180 ppm 

medication in drinking water 

during 10 days starting 11 days 

after infection 

Tylosin tartrate 50 mg/kg 

combined with Tiamulin 10 

mg/kg 

medication in drinking water 

during 10 days starting 14 days 

after infection 

Effects 

improvement of DWG and FCR 

improvement of lung lesions by 6- 

chloro analogue at 400 ppm only 

improvement of DWG and FCR 

lung lesions were not prevented 

M. hyopneumoniae could still be 

isolated 

improvement of DWG, clinical signs, 

lung lesions 

M. hyopneumoniae could still be 

isolated 

improvement of DWG, FCR, clinical 

signs, and macroscopic and microscopic 

lung lesions 

no beneficial effect on DWG, FCR, 

clinical signs, and macroscopic and 

microscopic lung lesions 

improvement of macroscopic lung 

lesions 

M. hyopneumoniae could not be 

isolated 

less frequently secondary bacterial 

infections 

Reference 

Hannan and Goodwin, 

1990 

Schuller et al., 1977 

Goodwin, 1979 

Kobisch and Sibelle, 

1982 

Ross and Cox, 1988 

Hannan et al., 1982 

DWG: daily weight gain; FCR: feed conversion ratio 

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Table 3: The effect of different antimicrobial regimens for treatment of Mycoplasma 

hyopneumoniae infections in herds clinically affected by enzootic pneumonia. 

Treatment 

Effects 

Reference 

Enrofloxacin 5 mg/kg 

Per os during 3 weeks starting after birth 

followed by parenteral treatment during 

3 weeks every 3 days starting in nursery 

unit followed by parenteral treatment 

during one week every 2 days 

prevention of clinical signs Frank, 1989 

Marbofloxacin 3 mg/kg 

IM during 3 days 

decrease of rectal temperature, 

improved DWG and clinical signs 

Thomas et al., 

2000 

Chlortetracycline 800 ppm 


Chlortetracycline 110 ppm 

feed medication for 1, 2, 3 or 4 months 

improvement of clinical signs and 

oxygen saturation 

no improvement of DWG, FCR and 

macroscopic lesions 

Ganter, 1995 

Ice et al., 1999 

Doxycycline 11 mg/kg 

feed medication during 8 days in the 

fattening unit 

improvement of DWG, incidence of 

diseased pigs and cure rate 

Bousquet et al., 

1998 

Lincomycin 5 mg/kg 

parenteral at day 1, 2, 3, and at day 39, 

40, 41 

Lincomycin 220 ppm 


improved DWG, FCR, and clinical signs 

no significant improvements 

Lukert and 

Mulkey, 1982 

Mateusen et al., 

2002 


feed medication during 10 days in the 

growing unit 


feed medication during 8 weeks in pigs 

from 30 to 70 kg 

Tiamulin 100 ppm combined with 

Chlortetracycline 300 ppm or 

Chlortetracycline 300 ppm alone 


improvement of DWG, clinical signs, 

macroscopic lung lesions, and mortality 

rate 

improvement of DWG, FCR 

no decrease in macroscopic lung lesions 

improvement of DWG, FCR, and 

no improvement of macroscopic lung 

lesions for the combined group 

improvement of DWG for the single 

group only 

Martineau, 1980 

Burch, 1984 

Burch et al., 1986 

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Tylosin 4 mg/kg or 

Lincomycin 5 mg/kg 

parenteral during 3 days after birth and 

for 3 days at weaning 

Tilmicosin 300 ppm in feed 

during 9 or 14 days 

Tilmicosin 200 ppm in feed 

during 3 weeks after weaning (days 34- 

55) and during 2 weeks in the nursery 

period (days 77-98) 

improved DWG with Tylosin 

no significant improvement of FCR and 

clinical signs with both antibiotics 

improved DWG, clinical signs 

less secondary bacteria 

Beneficial effects of DWG, FCR, 

mortality, and macroscopic lesions 

comparable to M. hyopneumoniae 

vaccination 

Kunesh, 1981 

Binder et al., 1993 

Mateusen et al., 

2001 

Tiamulin 200 ppm in feed 

Chlortetracycline 600 ppm in feed 

pulse medication: 

2 days treatment/2 weeks during the 

fattening period 


Oxytetracycline 300 ppm in feed 


2 or 3 days treatment/week during the 



Oxytetracycline 300 ppm in feed 


2 days treatment/week during the 



during 7 day at weaning and 

during 7 days at 4 months of age (and 

during 7 days at 6 months of age) 

lower prevalence of macroscopic lung 

lesions 

no influence on severity of macroscopic 

lung lesions 

improved DWG and FCR, prevalence 

and severity of lung lesions 

financial benefit 

improved DWG and mortality rate 

no improvement of FCR and severity of 

lung lesions 

Improved DWG and mortality rate, 

improvement of FCR and severity of 

lung lesions, financial benefit 

Le Grand and 

Kobisch, 1996 

Kavanagh, 1994 

Jouglar et al., 1993 

Stipkovits et al., 

2003 

DWG: daily weight gain; FCR: feed conversion ratio; IM: intramuscular 

ANTIMICROBIAL SUSCEPTIBILITY TESTING FOR MYCOPLASMA SPP. 

The Clinical and Laboratory Standards Institute issues no specific guidelines 

for susceptibility testing of Mycoplasma spp. Therefore, there has been little 

standardization of the procedures used to determine the minimal inhibitory 

concentration (MIC) of antimicrobials against these micro-organisms. This makes the 

comparison of MIC results between laboratories difficult. Standard broth and agar 

dilution methods used for susceptibility testing of other bacteria have been adapted for 

use with mycoplasmas. These methods, performed with appropriate controls, give 

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useful results for the treatment of mycoplasmal infections. Recently ‘Guidelines and 

recommendations for antimicrobial minimum inhibitory concentration (MIC) testing 

against veterinary mycoplasma species’ were proposed by the ‘International Research 

Programme on Comparative Mycoplasmology (IRPCM)’ (Hannan, 2000). No single 

standardized medium or pH can be indicated for mycoplasmas since growth 

requirements vary with the species. 

An inoculum of 10 4 to 10 5 colour changing units (CCU) per ml has been 

recommended. If available, a control strain with well established MIC ranges of the 

tested drugs should be included to validate the test. Finally, mandatory controls such 

as sterile growth medium and the control strain in drug-free medium are required. The 

choice of test procedure is influenced by several factors such as the number of strains 

to be tested, their growth titer in broth or agar, and the generation time of the species. 

Broth dilution and especially microbroth dilution techniques have been 

modified for mycoplasmas and are the most widely used. The method employs 

mycoplasmal media with increasing antibiotic concentrations, inoculated with a 

standardized number of micro-organisms in 96-well microtiter plates. Commercially 

available 96-well microtiter plates precoated with antimicrobials can be ordered and 

used with good results (Tanner and Wu, 1992; Tanner et al., 1993). Mycoplasma 

growth results in degradation of the metabolizable substrate such as glucose, arginin 

or urea, present in the medium with the resulting pH change visible as a colour change 

of a pH indicator. The MIC is defined as the lowest concentration of an antimicrobial 

agent that prevents a colour change at the time when the colour in the control without 

antibiotics has changed. The MIC of an antimicrobial agent for M. hyopneumoniae 

will only be available after 14 days of incubation. Turbidity or colour change in broth 

control indicates bacterial contamination. 

MICs of mycoplasmas growing more easily on solid agar, e.g. M. bovis 

(Francoz et al., 2005) and M. hominis (Waites et al., 1999), can also be determined 

using the E-test (AB BIODISK, Solna, Sweden). This test is based on the transfer of a 

continuous concentration gradient of an antimicrobial agent from a plastic strip into 

agar medium (Waites et al., 1997). 

No specific breakpoints for mycoplasmas are available, so it is preferable to 

merely report MICs. Some laboratories have adopted the breakpoints used for the 

interpretation of MICs for other bacteria (Bébéar and Bébéar, 2002). The 

development of antimicrobial resistance can be determined using the microbiological 

- 40 -


criterion. When a monomodal Gaussian distribution is found, no acquired resistance is 

present, but when the distribution is bimodal or multimodal a resistance mechanism 

can be supposed. Also when tailing, i.e. the loss of the normal Gaussian distribution, 

is seen, a resistance mechanism can be supposed (Butaye, 2000). 

IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE TO ANTIMICROBIALS USED 

IN PIG VETERINARY MEDICINE 

Few studies are available that document the in vitro susceptibilities of M. 

hyopneumoniae against antimicrobials used in swine practice to treat respiratory 

disorders. Since M. hyopneumoniae is a fastidious organism to isolate and the 

procedure is time consuming, only limited numbers of isolates were used for MIC 

determination. Broth is preferable and most frequently used (Table 4). 

Inconsistencies in results were seen in the susceptibility testing of 

tetracyclines. Williams (1978) found chlortetracycline to be less active than 

oxytetracycline and doxycycline, Etheridge et al. (1979) found a wide variation in the 

MICs (1.6 – 25 µg/ml) for chlortetracycline and Yamamoto and Koshimizu (1984) 

reported high MICs (≥ 40 µg/ml) for chlortetracycline. Inamoto et al. (1994) observed 

an increase in resistance to both chlortetracycline and oxytetracycline occurring in 

Japanese M. hyopneumoniae field strains isolated between 1970 and 1990. MICs of 

unstable antimicrobials like chlortetracycline and valnemulin are unreliable since 

MIC determination of M. hyopneumoniae can take up to 14 days. 

In contrast to most studies reporting the lack of acquired resistance in M. 

hyopneumoniae, Wu et al. (1997) found several resistant isolates. Of the 14 isolates 

tested, 13 were resistant against tylosin, although 10 were susceptible to tilmicosin. 

One isolate was resistant to lincomycin, apramycin and enrofloxacin, two against 

spectinomycin. All isolates were susceptible to tetracycline and gentamicin. Because 

of inconsistency in the resistance profile against macrolides and the fact that the M. 

hyopneumoniae isolates were incubated for only 3 days, the results should be 

interpreted with caution and are therefore not included in Table 2. 

In conclusion, despite some reports on resistance against tetracyclines, 

acquired resistance in M. hyopneumoniae does not seem to constitute a major problem 

to date. Genotypical confirmation of resistance has not been reported for M. 

hyopneumoniae. 

- 41 -


- 42 -


- 43 -


ANTIMICROBIAL RESISTANCE IN MYCOPLASMA SPP. 

Intrinsic, innate or natural resistance is defined as the relative insensitivity of 

all members of a bacterial species or genus against an antimicrobial agent. Some 

intrinsic resistances can be related to only one Mycoplasma species, while others can 

be related to all members of the class of Mollicutes. By contrast, resistance may be 

acquired by some strains within a species usually susceptible to the antimicrobial 

agent under consideration. 

Similar biochemical mechanisms may be responsible for intrinsic and acquired 

resistances, e.g. target modification, decreased uptake or enzymatic modification of 

the antimicrobial agent. 

Intrinsic resistance related to the class Mollicutes 

Mollicutes lack a cell wall and are therefore resistant to all β-lactam 

antibiotics, like penicillin, ampicillin, amoxicillin, methicillin and cephalosporins. 

Also other antimicrobials targeting the cell wall, like glycopeptides (vancomycin, 

teicoplanin and avoparcin) are of no use in the treatment of mycoplasma infections. In 

addition, mycoplasmas are also resistant to polymyxins, sulfonamides, trimethoprim, 

naladixic acid and rifampin. 

Intrinsic resistance related to the Mycoplasma species 

Innate resistance related to specific Mycoplasma species concerns mainly the 

macrolides. As described above, macrolides are classified into three subgroups 

according to their lactone ring sizes (14-, 15- and 16-membered lactone ring). M. 

hyopneumoniae, M. flocculare, M. bovis, M. pulmonis and M. fermentans are 

insensitive to the 14-membered lactone ring macrolides, erythromycin and 

oleandomycin (Williams, 1978; Tanner et al., 1993; Chambaud et al., 2001; Bébéar 

and Bébéar, 2002; Francoz et al., 2005), whereas M. pneumoniae and M. genitalium 

are susceptible (Bébéar et al., 2000; Kenny and Cartwright, 2001). The genetic basis 

of this innate resistance has been elucidated. Macrolides bind to the bacterial 

ribosome and affect its peptidyltransferase activity in the V domain of 23S rRNA. In 

- 44 -


erythromycin and oleandomycin resistant mycoplasmas, a G →A transition at position 

2057 is found in the sequence of the 23S rRNA. 

Acquired antimicrobial resistance in Mycoplasma spp. 

Generally speaking, there are three main mechanisms of acquired 

antimicrobial resistance in mycoplasmas: active efflux mechanisms, enzymatic 

modification of the drug or alteration in the drug target site. Mycoplasmas are 

characterized by high mutation frequencies due to the limited amount of genetic 

information dedicated to DNA repair systems (Razin et al., 1998). 

1. Tetracyclines 

Acquired resistance to tetracyclines has been documented in human 

mycoplasmas of the urogenital tract: M. hominis and Ureaplasma urealyticum (Kenny 

and Cartwright, 1994; Waites et al., 1997). In veterinary medicine, tetracycline 

resistance has been described in M. bovis and M. hyopneumoniae (Inamoto et al., 

1994; Thomas et al., 2003). Resistance in M. hominis and Ureaplasma spp. to 

tetracycline has been associated with the presence of the tetM determinant (Roberts et 

al., 1985; Blanchard et al., 1992). Blanchard et al. (1992) reported that all of the tetMpositive 

M. hominis isolates were sensitive to doxycycline, indicating that tetM does 

not necessarily confer resistance to this antibiotic. Recent research supports the 

assumption that tetracycline resistance in mollicutes can also be mediated by other 

genes than tetM (Taraskina et al., 2002). In veterinary mycoplasmas, no resistance 

mechanisms have currently been described. 

2. Macrolides and lincosamides 

Acquired resistance to macrolides is not well documented in human (M. 

pneumoniae, M. hominis and U. urealyticum) and animal (M. bovis and M. 

gallisepticum) mycoplasmas, but seems to increase considerably during the last years 

(Samra et al., 2002; Thomas et al., 2003; Francoz et al., 2005; Morozumi et al., 

2005). In vitro selected resistant Mycoplasma strains showed mutations in the domain 

V of the 23S rRNA. Positions 2058, 2059 (E. coli numbering) seem to be hot spots for 

- 45 -


macrolide resistance. This is in agreement with the situation in other bacteria (Lucier 

et al., 1995; Alvarez-Elcoro and Enzler, 1999; Pereyre et al, 2004; Wu et al., 2005). 

In addition, position 2062 was determined as a position responsible for resistance 

against 16-lactone ring macrolides (Furneri et al., 2001). Recently, mutations in the 

ribosomal proteins L4 and L22 have been associated with increased MIC-values in 

Gram-negative bacteria and Streptococcus spp. (Franceschi et al., 2004; Roberts, 

2004; Prunier et al., 2005). Similar mutations were found in M. pneumoniae after in 

vitro selection (Pereyre et al., 2004) but not in clinical isolates. The resistance 

mechanism in veterinary important mycoplasmas has not yet been elucidated but it is 

probably similar to that in human mycoplasmas. 

3. Pleuromutilins 

Development of resistance against pleuromutilins in animal-associated 

mycoplasmas isolated from clinical specimens has not yet been described. One 

publication (Gautier-Bouchardon et al., 2002) reports the development of resistance in 

some M. iowae isolates after repeated passages in vitro in the presence of tiamulin, 

but the mechanism was not described. 

4. Fluoroquinolones 

Two fluoroquinolone resistance mechanisms have been described: 1) mutation 

in the four target genes, gyrA, gyrB, parC and parE and 2) reduction in the level of 

quinolone accumulation inside the cells either by efflux or lack of penetration 

(Hooper, 1999). 

In human medicine, acquired resistance has only been reported for genital 

mycoplasmas. The veterinary important mycoplasmas M. gallisepticum (Reinhardt et 

al., 2002) and M. bovis (Thomas et al., 2003) have been shown to be able to develop 

resistance against fluoroquinolones in vivo. Resistance is based on stepwise alterations 

in the four target genes but as in other Gram-positive bacteria, mainly gyrA and parC 

are involved (Chen and Lo, 2003). The existence of efflux mechanisms increasing the 

MIC has only been demonstrated in M. hominis (Raherison et al., 2002). 

- 46 -


5. Florfenicol 

No resistance against florfenicol has been described in animal-associated 

mycoplasmas. 

6. Aminoglycosides and aminocyclitols 

Acquired resistance to aminoglycosides has been described for M. bovis 

(Thomas et al., 2003) and M. hyorhinis (Idowu et al., 2003). Resistance mechanisms 

in other bacteria include mutations in the ribosome target, presence of efflux 

mechanisms, or the presence of aminoglycoside-modifying enzymes (aminoglycoside 

phosphotransferases, aminoglycoside nucleotidyltransferases and aminoglycoside 

acetyltransferases) (Kotra et al., 2000). Stepwise selections of Mycoplasma strains 

grown in the presence of aminoglycosides or aminocyclitols result in resistance 

against aminoglycosides, but the resistance mechanism has not been elucidated 

(Hannan, 1995). 

PREVENTIVE MEDICATION VERSUS VACCINATION 

The use and efficacy of either vaccination or preventive (strategic) medication 

is frequently discussed among swine veterinarians and both strategies have 

advantages and disadvantages. Advantages of antimicrobial prevention strategies are 

the flexibility, antimicrobials are often effective against several (respiratory) 

pathogens and the administration is less labour-intensive since group treatments are 

mainly accomplished by in-feed or in-water medication. Vaccination on the other 

hand, does not select for antimicrobial resistance in pathogenic bacteria and in 

bacteria belonging to the microbiota of the animal. It also avoids risks for 

antimicrobial residues in slaughter pigs. While for antimicrobial treatment, an 

immediate effect at herd level can be expected, the effect of vaccination of young 

piglets will only be evident if it is continued during approximately 6 months. It is not 

warranted to interrupt vaccination during periods of low risk for disease outbreaks or 

during periods of poor market conditions (Maes et al., 2003). Vaccination against M. 

hyopneumoniae has as additional effect that secondary bacterial infections 

- 47 -


(Pasteurella multocida, Actinobacillus pleuropneumoniae) less frequently occur 

under field conditions (Kobisch et al., 1993; Sørensen et al., 1997). 

Neither vaccination nor preventive medication can prevent infection and 

adherence of M. hyopneumoniae to the ciliated cells of the respiratory tract of treated 

or vaccinated pigs has been demonstrated in several studies (Hannan et al., 1982; 

Kubo et al., 1990; Le Grand and Kobisch 1996, Schatzmann et al., 1996, Mateusen et 

al., 2002). 

ERADICATION OF M. HYOPNEUMONIAE 

Eradication of enzootic pneumonia would result in serious savings each year 

and in improvement of the health and welfare of the pigs. 

A depopulation and restocking programme has been proposed and conducted 

in 1960 by the Pig Health Control Organisation (Goodwin and Wittlestone, 1960) and 

eradication of herds in the UK was described by Goodwin and Wittlestone (1967). 

This programme was expensive and reinfections occurred in 20% of the herds. 

Now, eradication of M. hyopneumoniae is mainly applied in Denmark, Finland 

(Heinonen et al., 1999) and Switzerland (Zimmerman et al., 1989; Zimmerman, 

1990). In Switzerland, a total and partial sanitation programme was set up 

(Zimmermann et al., 1989). The total sanitation programme involved complete 

emptying of the animal facilities by selling or culling all animals. The partial 

sanitation programme included a piglet and gilt free (< 10 months) interval and the 

temporary feeding of a medicated diet during 10-14 days. The feed contained either 

tiamulin (6 mg/kg body weight (BW)) or a combination of chlortetracycline (20 

mg/kg BW), tylosin (4 mg/kg BW) and sulfadimidin (30 mg/kg BW). 

Another method used mainly in the US is ‘medicated early weaning (MEW)’ 

wherein intensive medication of the sow during late gestation and immediately 

following parturition and of the newborn piglets is used to derive pigs free of M. 

hyopneumoniae. Piglets are weaned at 5 days of age, placed in an isolated nursery on 

a separate site and medicated for the first 10 days after weaning (Alexander et al., 

1980). Modifications of the MEW programme, based on postponing weaning until 21- 

25 days of age, keeping sows and nursery pigs at the same site, vaccination strategies 

and/or combined with medication of the sows and medications of the piglets before 

and after weaning were described and evaluated by Clark et al. (1994). It was 

- 48 -


concluded that isolating the pigs was as effective as medication and vaccination 

protocols in controlling the transmission of the pathogens investigated, including M. 

hyopneumoniae, Bordetella bronchiseptica and Pasteurella multocida. M. 

hyopneumoniae was not detected when any MEW procedure was used. 

Antimicrobials used for eradication programmes are preferably 

mycoplasmacidal, like the newer fluoroquinolones, ciprofloxacin and enrofloxacin 

(Hannan et al., 1989) and aminoglycosides. Tiamulin is most frequently utilized to 

sustain eradication programmes (Mészáros et al., 1985). 

Although several attempts have been made to eradicate M. hyopneumoniae 

from a herd, reinfection frequently occurs (2.6-10% per year) (Keller, 1987, 

Whittlestone, 1990; Hege et al., 2002). A prominent explanation for this finding is 

that neighbouring herds are not free of M. hyopneumoniae (Goodwin, 1985; Jorsal 

and Thomsen, 1988, Wallgren et al., 1993; Hege et al., 2002). M. hyopneumoniae is 

able to spread over a distance of 3.2 km (Goodwin, 1985). Other statistically 

significant risk factors for the reinfection as determined by Hege et al. (2002) were 

'finishing farm', 'large mixed breeding-finishing farm', and 'parking site for pig 

transport vehicles close to the farm'. A protective factor was the purchase of pigs from 

one supplier per batch. Another explanation might be the technique used to 

investigate the infection status of M. hyopneumoniae of purchased pigs. Mainly 

ELISA techniques on serum or colostrum samples are used. ELISA has proved to be a 

very useful technique but recently it was demonstrated that the pigs can be colonized 

by low numbers of M. hyopneumoniae organisms in the lung without showing 

seroconversion (Thacker, 2004). 

Based on the cited studies, it can be stated that vaccination or antimicrobial 

treatment can indeed reduce the number of organisms in the lungs but these measures 

do not guarantee the absence of M. hyopneumoniae. MEW contributes to prevent 

spread of M. hyopneumoniae from sow to offspring. 

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1.2 AIMS

Mycoplasma hyopneumoniae is the primary cause of enzootic pneumonia, a 

disease which is responsible for major economic losses in the pig industry worldwide. 

In several studies it was shown that environmental factors influence the clinical 

course of the disease and the extent of economic losses in infected herds. Variation in 

the clinical course of the disease may, however, also be due to differences in virulence 

between different M. hyopneumoniae strains, as has been demonstrated for several 

other bacterial pathogens. No studies dealing with this subject are available in the 

literature. The first general aim of this thesis was therefore to evaluate the virulence of 

M. hyopneumoniae field isolates. 

Antimicrobials are often used in the field to treat M. hyopneumoniae infected 

animals. As stipulated in the review of the literature, very few data are available on 

the susceptibility of M. hyopneumoniae field isolates. This is mainly due to the 

fastidious nature of this micro-organism that is extremely hard to isolate and to 

cultivate, making it difficult to obtain field isolates and to test their antimicrobial 

susceptibility in vitro. A second general aim of this thesis was to determine the 

susceptibility of M. hyopneumoniae field isolates to different antimicrobials. 

The specific aims of this study were: 

- To compare patterns of M. hyopneumoniae infections in farrow-to-finish 

pig herds with diverging disease-course, 

- to evaluate the virulence of M. hyopneumoniae field isolates in an 

experimental infection model, 

- to determine in vitro susceptibility of M. hyopneumoniae field isolates to 

antimicrobial agents, 

- to characterize the genetic mechanisms of acquired resistance to 

macrolides and fluoroquinolones in M. hyopneumoniae, 

- to determine the efficacy of in feed medication with tylosin for treatment 

of an experimental M. hyopneumoniae infection. 

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2 EXPERIMENTAL STUDIES 

- 63 -

2.1 VIRULENCE OF MYCOPLASMA HYOPNEUMONIAE 

ISOLATES 

- 65 -

2.1.1 PATTERNS OF MYCOPLASMA HYOPNEUMONIAE 

INFECTIONS IN BELGIAN FARROW-TO-FINISH PIG HERDS WITH 

DIVERGING DISEASE-COURSE 

Modified from: 

PATTERNS OF MYCOPLASMA HYOPNEUMONIAE INFECTIONS IN BELGIAN FARROW-TO- 

FINISH PIG HERDS WITH DIVERGING DISEASE-COURSE 

J. VICCA, D. MAES, L. THERMOTE, J. PEETERS, F. HAESEBROUCK & A. DE KRUIF 

JOURNAL OF VETERINARY MEDICINE, SERIES B 2002, 49, 349-353 

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2.1.1 PATTERNS OF M. HYOPNEUMONIAE INFECTIONS IN FARROW-TO-FINISH PIG HERDS 

SUMMARY 

Patterns of Mycoplasma hyopneumoniae infections were investigated in 5 

clinically infected herds and in 5 herds subclinically infected with M. hyopneumoniae. 

In the clinically infected herds, housing and management conditions were good 

whereas these conditions were poor in the subclinically infected herds. In each herd, 

serumantibodies against M. hyopneumoniae were detected in pigs of different ages 

and nasal swabs were taken for M. hyopneumoniae detection using nested PCR 

(nPCR). The percentage of seropositive pigs in the clinically infected herds increased 

from 8% in pigs of 9 weeks to 52% in pigs of 18 weeks and seroconversion was most 

shown between 12 and 15 weeks. In the subclinically infected herds, the percentages 

increased from 2% to 24% and most of the pigs became seropositive between 15 and 

18 weeks. The percentage of nPCR positive pigs at 6 weeks was 16% and 0% in the 

clinically and subclinically infected herds, respectively. The results demonstrate that 

the seroprevalences were higher in the clinically infected herds and that most of the 

pigs became infected with M. hyopneumoniae at a younger age. It can be concluded 

that additional factors different from housing and management, like differences 

among M. hyopneumoniae isolates, may determine the infection pattern of M. 

hyopneumoniae and the clinical course of the infection. 

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INTRODUCTION 

Enzootic pneumonia in pigs, caused by M. hyopneumoniae as primary agent, 

is a chronic respiratory disease that causes major economic losses to the pig industry. 

Infections with M. hyopneumoniae are very common worldwide, especially in 

countries with intensive production systems. Epidemiological studies showed that 

more than 90% of the swine farms in Belgium are infected with M. hyopneumoniae 

(Maes et al., 1999a; 2000). The disease is mainly characterized by chronic coughing 

and reduced performance in grow-finishing pigs, although many infections with M. 

hyopneumoniae occur without overt clinical symptoms (Ross, 1999). The control of 

the disease can be accomplished in a number of different ways. First of all, the 

housing conditions of the pigs must be optimal and they must be appropriate 

according to the age of the pigs. Management practices that reduce the spread of M. 

hyopneumoniae infections e.g. optimal stocking density, no mixing of pigs, all-in/allout 

(AIAO) production, must be adopted. Additional control measures such as the 

preventive use of antimicrobials during periods of risk and/or vaccination against M. 

hyopneumoniae have been used in many swine farms. The latter measures are 

generally effective in reducing the severity and economic losses of the disease and in 

improving the performance of the pigs (Clark et al., 1991; Legrand and Kobisch, 

1996). 

In some farms, however, these measures, applied either individually or 

collectively, do not always control M. hyopneumoniae infections effectively. Farms 

with good housing conditions and management practices may suffer from clinical 

disease whereas in some infected farms with poor environmental conditions, M. 

hyopneumoniae infections do not cause obvious clinical disease. Similarly, 

medication and vaccination do not consistently provide the expected or desired level 

of control against M. hyopneumoniae infections (Mateusen et al., 2002). The 

beneficial effects of vaccination against M. hyopneumoniae vary from farm to farm 

(Morrow et al., 1994; Maes et al., 1999b) and the results cannot consistently be 

predicted beforehand. Therefore, it can be expected that besides housing and 

management conditions, other factors e.g. differences in M. hyopneumoniae isolates 

(Frey et al., 1992; Artiushin and Minion, 1996) may also determine the clinical course 

and infection pattern of M. hyopneumoniae. 

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The present field study aimed to investigate the infection pattern of M. 

hyopneumoniae in farms with either good or poor housing and management 

conditions. According to previous studies (Yagihashi et al., 1993; Horst et al., 1997), 

one should expect a higher prevalence and earlier infection of M. hyopneumoniae in 

the herds with poor housing and management. In addition, one should expect a higher 

chance for clinical disease in these farms (Pointon et al., 1985; Christensen et al., 

1999). However, we intentionally selected 5 farms with poor housing and 

management that were subclinically infected with M. hyopneumoniae, and 5 farms 

with good housing and management that were clinically infected with M. 

hyopneumoniae. The hypothesis was that, if the prevalence of M. hyopneumoniae is 

higher in the clinically infected herds with good housing and management conditions 

and if pigs are infected at a younger age in these herds, then additional factors other 

than housing and management also determine the course of the infection within these 

swine farms. The infection pattern was assessed using serology and nested PCR 

(nPCR) on nasal swabs of pigs of different age groups. 

MATERIALS AND METHODS 

Selection and description of the study farms 

Ten farrow-to-finish pig herds, located in 4 different provinces of Flanders 

(Belgium), participated in the study (Table 1). They were selected by contacting the 

herd health veterinarian. The investigator visited all herds once. All the herds had at 

least 100 sows and they were infected with M. hyopneumoniae as demonstrated by 

previous laboratory data. Herds that had used M. hyopneumoniae vaccination in the 

past 3 years were excluded. The breeding population mainly consisted of hybrid sows 

that were either purchased or produced by on-farm selection. The sows were 

inseminated with semen from Piétrain boars for the production of slaughter pigs. The 

parity distribution of the sows was very similar between the selected farms. On 

average, sows had 3.5 to 4.5 litters before they were culled. In all the herds, pigs were 

weaned when they were about 4 weeks old. At that time, they were transferred to the 

nursery unit in which they were housed until 10-12 weeks of age. Thereafter, they 

were moved to the grow-finishing unit in which they remained until slaughter age (6- 

7 months). A fully slatted floor was present in the grow-finishing units of all farms. 

- 71 -


Table 1: Some important characteristics of the 10 selected herds infected with 

Mycoplasma hyopneumoniae that participated in the study 

Herd 

Infection 

Pig density in the 

Herd size 

Purchase of 

Production system 

status a 

Municipality 

(number of 

gilts 

(# of pigs/km²) 

sows) 

1 0 1123 120 No AIAO in N, 

Continuous in G-F b 

2 0 1363 220 Yes AIAO in N, 

Continuous in G-F 

3 0 845 120 Yes Continuous 

4 0 393 120 No Continuous 

5 0 1413 200 No Continuous 

6 1 1363 240 Yes AIAO 

7 1 1472 100 Yes AIAO 

8 1 1123 100 Yes AIAO 

9 1 1123 200 No AIAO 

10 1 875 300 No AIAO 

a 

b 

0 subclinically infected herds; 1 clinically infected herds 

AIAO all-in/all-out; N nursery unit; G-F grow-finishing unit 

In 5 of the 10 herds, the housing conditions and management practices were 

optimal although the herds were clinically infected with M. hyopneumoniae for at 

least one year. Good housing conditions implied the presence of 

compartmentalization, indirect air-inlets so that the incoming air is warmed before 

reaching the pig area, and programmes to maintain appropriate temperatures in the 

rooms according to the age of the pigs. Compartmentalization is the subdivision of a 

barn into rooms with a capacity of about 100-150 pigs and with its own ventilation 

system. The management practices in these farms included the use of all-in/all-out 

(AIAO) production in the nursery and grow-finishing units, maintaining optimal 

stocking densities of the pigs, and practicing strict internal and external biosecurity 

measures. According to the swine producer, the herd health veterinarian and the 

investigator, the herds were all clinically infected with M. hyopneumoniae. This was 

evidenced by the presence of chronic coughing in nursery and grow-finishing pigs, 

and by reduced production parameters in grow-finishing pigs (reduced average daily 

- 72 -


weight gain, heterogeneous growth, and poor feed conversion) compared to similar 

herds without chronic coughing. The clinical course of the disease and data from 

diagnostic laboratories indicated that the problems were not primarily due to 

infections with other respiratory pathogens such as Actinobacillus pleuropneumoniae 

or swine influenza viruses. 

In the 5 remaining herds, the housing conditions and management practices 

were poor although the herds were not clinically infected with M. hyopneumoniae. 

The swine barns were old and worn out, there was no compartmentalization, no 

indirect air-inlet, and no specific programmes to maintain appropriate temperatures in 

the rooms. A continuous production system was practised or AIAO was used only in 

the farrowing and nursery unit, the stocking density in grow-finishing pigs was too 

high (


Nasal samples and nPCR for M. hyopneumoniae 

Nasal swabs were collected in each herd from pigs belonging to 4 different 

age groups namely 6, 9, 12 and 15 weeks. Five pigs per age category were selected. 

The pigs of the youngest age group were selected randomly at pen-level; those of the 

other age groups were randomly selected out of the pigs that were blood sampled. The 

pigs of 6 and 9 weeks old were housed in the nursery unit, those of 12 and 15 weeks 

in the grow-finishing unit. None of the sampled pigs had received antimicrobials in 

the last 4 weeks. 

The nasal swabs were 15 cm long and they were inserted into the nostrils as deeply as 

possible. The swabs were transported in 700 µL PBS with 0.1% Triton x-100, and 

they were vortexed and discarded upon arrival in the laboratory. The transport 

medium was heated at 100°C for 5 min and stored frozen at –25°C until PCR was 

performed. Five µL of undiluted sample and 5 µL of 10-fold diluted sample were used 

in the nPCR, which was performed as described by Stärk et al. (1998). PCR products 

were analyzed by electrophoresis on 1% agarose gels in TBE buffer and visualized 

under UV illumination after staining in Ethidium Bromide. 

Statistical analysis 

The percentage of seropositive pigs and the percentage of nPCR positive pigs 

in each age category were compared between the clinically and the subclinically 

infected herds by means of Fisher's Exact Tests, performed on the raw numeric data. 

The average OD-values of the different age groups were compared between the 

clinically and the subclinically infected herds using two-sample t-tests. Variables 

were considered to be significant when P-values were lower than 0.05 (two-sided). 

Data were analyzed using the statistical package SPSS 10.0 for Windows (SPSS 10, 

SPSS Inc. Illinois 60611 USA 1999). 

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RESULTS 

Blood samples and serology for M. hyopneumoniae 

The percentage of seropositive pigs in the clinically infected herds increased 

from 8% in pigs of 9 weeks to 52% in pigs of 18 weeks, whereas in the subclinically 

infected herds the percentages in the same age categories increased from 2% to 24% 

(Table 2). At 15 and 18 weeks, the percentage of seropositive pigs was significantly 

higher in the clinically infected herds than in the subclinically infected herds 

(P


nPCR positive pigs was almost similar in the clinically (8 and 12%, respectively) and 

subclinically (12 and 12%, respectively) infected herds (P>0.05). 

Table 2. The percentage of seropositive pigs a and the average OD-values b in the 

different age categories of 10 farrow-to-finish pig herds that were either clinically (n=5) 

or subclinically (n=5) infected with Mycoplasma hyopneumoniae 

% of seropositive pigs (min.-max.) Average OD-value (SD) 

Age 

(Weeks) 

Clinically 

infected herds 

Subclinically 


P-value 

Clinically 


Subclinically 


P-value 

9 8 (0-40) 2 (0-10) 0.362 79.1 (18.27) 82.3 (21.79) 0.088 

12 8 (0-40) 0 (0- 0) 0.117 79.8 (16.80) 83.7 (09.02) 0.157 

15 20 (0-70) 2 (0-10) 0.008 69.7 (24.36) 83.5 (13.36) 0.013 

18 52 (0-100) 24 (0-40) 0.007 53.0 (26.71) 64.7 (22.63) 0.021 

a 

b 

10 pigs per age category were randomly selected at pen-level in each herd 

Average OD-values (%) were based on all investigated pigs per age category 

- 76 -


Table 3. The results of the nested PCR (nPCR) on nasal swabs from pigs of different age 

categories in 10 farrow-to-finish pig herds that were clinically (n=5) or subclinically 

(n=5) infected with Mycoplasma hyopneumoniae 

Age category (weeks) 

% (min.-max.) of positive pigs a, b 

Clinically infected herds 

Subclinically infected herds 

6 16 (0-40) 0 (0-0) 

9 24 (0-40) 12 (0-20) 

12 8 (0-20) 12 (0-40) 

15 12 (0-40) 12 (0-40) 

a 

b 

5 pigs per age category were randomly selected in each herd 

there were no significant differences between both types of herds (P>0.05) 

DISCUSSION 

The study assessed the patterns of M. hyopneumoniae infections in farms with 

good housing and management conditions that were clinically infected with M. 

hyopneumoniae, and in farms with poor housing and management conditions that 

were subclinically infected with M. hyopneumoniae. The hypothesis was that, if the 

prevalence of M. hyopneumoniae is higher in the clinically infected herds and if pigs 

are infected at a younger age in these herds, then additional factors different from the 

housing and management e.g. differences among circulating M. hyopneumoniae 

isolates, are associated with the course of the infection within swine farms. A 

difference in infection pattern due to a different parity of the sows in the herds is very 

unlikely since all herds had a comparable parity distribution. Although it cannot be 

ruled out, it is also unlikely that infections with other pathogens like PRRSV or 

Circovirus type 2 may have accounted for the different M. hyopneumoniae infection 

pattern in the selected farms since almost all Belgian herds are infected with PRRSV 

(Maes et al., 1997) and Circovirus type2 (Labarque et al., 2001). It is generally 

accepted that the housing and management conditions play a key-role in the spread 

and clinical course of M. hyopneumoniae infections in swine farms (Clark et al., 

1991; Ross, 1999). In this respect, one should have expected a more intensive spread 

in the herds with poor housing and management conditions. The results of the 

- 77 -


serology and nPCR however, demonstrated that this was not the case. Moreover, the 

seroprevalence of M. hyopneumoniae was higher and most of the pigs became 

infected with M. hyopneumoniae at a younger age in the clinically infected herds with 

good housing and management than in the subclinically infected herds with poor 

housing and management. 

The results of the present study do not underestimate the importance of 

housing and management factors in the transmission of M. hyopneumoniae infections 

in swine farms (Ross, 1999). The results demonstrate that the presence of poor 

housing and management in M. hyopneumoniae infected herds does not automatically 

imply that the clinical symptoms associated with M. hyopneumoniae infections will be 

severe, or that the spread of M. hyopneumoniae infections will be more intensive than 

in other farms. Conversely, the results also demonstrate that good housing and 

management practices do not always result in a subclinical course of M. 

hyopneumoniae infection. It can thus be deduced that in addition to housing and 

management, other factors are involved in the transmission of M. hyopneumoniae 

infections in swine farms. These factors are currently not yet fully understood, but it is 

reasonable to assume that differences among M. hyopneumoniae isolates concerning 

the antigenic, pathogenic and genetic characteristics may account for the different 

infection patterns in both types of farms. Some studies have shown some evidence for 

differences among M. hyopneumoniae isolates (Frey et al., 1992; Artiushin and 

Minion, 1996). Further research is currently performed in our laboratory to investigate 

the characteristics of the M. hyopneumoniae isolates from these farms. 

Although specific criteria have been used to select both groups of farms, the 

individual farms belonging to either the clinically or subclinically infected groups 

were not identical. The housing and management factors that are important for the 

respiratory health status of the pigs are very diverse and encompass many different 

aspects (Christensen et al., 1999). In this respect, it is difficult to select a group of 

farms with all exactly the same housing and management characteristics. However, in 

the present study, the most important factors of the housing and management that are 

known to be associated with enzootic pneumonia or with respiratory disease were 

considered for the selection of the farms. Likewise, it is hard to find farms with 

identical clinical symptoms of enzootic pneumonia in all age groups or in which 

clinical symptoms of enzootic pneumonia are totally absent. Important symptoms of 

enzootic pneumonia namely chronic coughing and reduced performance in grow- 

- 78 -


finishing pigs (Ross, 1999) have been considered for the selection procedure, and 

other respiratory diseases have been excluded. Thus, the farms belonging to the same 

group in the present study were not identical with respect to housing, management 

and clinical symptoms of M. hyopneumoniae infection, but they were very alike 

concerning these factors. 

The infection pattern of M. hyopneumoniae in the farms was assessed using 

serology and nPCR on nasal swabs of pigs of different age groups. Both diagnostic 

techniques have been shown to be valuable tools in profiling herds for M. 

hyopneumoniae infection (Calsamiglia et al., 1999). Serological testing is easy to 

perform and the DAKO ® Mh ELISA has a high sensitivity and specificity to detect 

serum antibodies following M. hyopneumoniae infection (Sørensen et al., 1992; 

1997). However, the serological response following M. hyopneumoniae infection is 

variable and the time-span between infection and seroconversion can take 3 to 8 

weeks (Nicolet et al., 1990; Kobisch et al., 1993; Morris et al., 1995). In addition, 

there is no correlation between the titer of serum antibodies against M. 

hyopneumoniae and the degree of protection against infection (Murphy et al., 1993; 

Yagihashi et al., 1993). Testing with nPCR on nasal swabs is a direct measure for 

detection of presence of nucleic acid of M. hyopneumoniae organisms. Consequently, 

a positive result does not automatically mean that this pig is actively shedding viable 

M. hyopneumoniae organisms. False negative results may arise if only negligible 

quantities of M. hyopneumoniae organisms are present in the nose, and/or if the nasal 

swab contains a lot of cellular debris that can inhibit the PCR (Stärk et al., 1992). 

The results of the nPCR in the present study corroborate with the serological 

results since the number of positive pigs in both tests was higher in the clinically 

infected herds and since both tests demonstrated that pigs became infected at a 

younger age in the clinically infected herds. However, the infection can be detected 

earlier with nPCR than with serology because there was a higher proportion of 

nursery pigs that was positive using nPCR testing compared to serology. This finding 

corroborates with results of previous studies (Calsamiglia et al., 1999) and it indicates 

that nPCR testing is more appropriate than serology to detect M. hyopneumoniae 

infection in young pigs at the farm level. In the clinically infected herds, 16% of the 

pigs at 6 weeks were already positive using nPCR. This means that they became 

- 79 -


infected at the beginning of the nursery period or possibly during the farrowing 

period. It also indicates that good housing and management do not guarantee per se 

that the occurrence of M. hyopneumoniae infection will be delayed until the growfinishing 

unit. No pigs younger than 9 weeks were blood sampled to avoid possible 

interference with maternal antibodies. Morris et al. (1994) showed that maternal 

antibodies against M. hyopneumoniae can persist as long as 9 weeks in pigs derived 

from sows with very high titres of serum antibodies. Although it cannot be ruled out 

with certainty, it is unlikely that the positive ELISA titres in a few pigs of 9 weeks 

were due to maternal antibodies. The number of seropositive pigs gradually increased 

towards the end of the finishing period. This reflects the chronic nature of M. 

hyopneumoniae infections (Ross, 1999). Even within clinically or subclinically 

infected herds, individual pigs started seroconverting at different ages (data not 

shown). In this respect, it is difficult to assess ‘the’ age of infection in a farm. It may 

be more relevant to determine the age at which most of the pigs became infected. 

CONCLUSION 

Even with a limited number of samples and the combination of nPCR on nasal 

swabs and serology, it was possible to assess the dynamics of M. hyopneumoniae 

infection in the selected farms and to elucidate differences in infection patterns 

between the selected clinically and subclinically infected herds. More studies also 

including subclinically infected farms with good housing and management and 

clinically infected farms with poor housing and management are needed to confirm 

the present findings. The results of this study document that additional factors 

different from the investigated housing and management conditions may determine 

the infection pattern of M. hyopneumoniae and the clinical course of the infection. 

Further research will focus on possible mechanisms that can explain the different 

infection patterns and on possible differences among M. hyopneumoniae isolates from 

these farms. 

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ACKNOWLEDGEMENTS 

This research was financially supported by grant S-5940 (section II) of the 

Belgian Ministry of Small Enterprises, Trader and Agriculture, Research Foundation 

(Brussels). The swine producers and herd health veterinarians are acknowledged for 

their participation in this study. 

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hyopneumoniae field isolates demonstrates genetic heterogeneity. International Journal of 

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2. Calsamiglia, M., Pijoan, C. & Bosch, G. (1999). Profiling Mycoplasma hyopneumoniae in 

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ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. Veterinary Microbiology 30, 

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6. Frey J., Haldimann, A. & Nicolet, J. (1992). Chromosomal heterogeneity of various 

Mycoplasma hyopneumoniae field strains. International Journal of Systemic Bacteriology 42, 

275-280. 

7. Horst, I., Lindner, A., Krüger, M., Gindele, H. & Sting, R. (1997). Verbreitung der 

Mycoplasma hyopneumoniae infektion in Deutschland. Schluβfolgerungen für die Bekämpfung 

der Enzootischen Pneumonie der Schweine. Tierärztliche Umschau 52, 508-514. 

8. Kobisch, M., Blanchard, B. & Le Potier, M.F. (1993). Mycoplasma hyopneumoniae infection 

in pigs: duration of the disease and resistance to reinfection. Veterinary Research 24, 67-77. 

9. Labarque, G.G., Nauwynck, H.J., Mesu, A.P. & Pensaert, M.B. (2001). Seroprevalence of 

porcine circovirus types 1 and 2 in the Belgian pig population. The Veterinary Quarterly 22, 

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10. LeGrand, A. & Kobisch, M. (1996). Comparison of the use of a vaccine and sequential 

antibiotic treatment in a herd infected with Mycoplasma hyopneumoniae. Veterinary Medicine 

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(1997). Descriptive epidemiological aspects of the seroprevalence of five respiratory disease 

agents in slaughter pigs from fattening pig herds. In: Proc. 8 th ISVEE Congress Paris France, 

05.B.19. 


(1999a). Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine 

Influenza viruses and Aujeszky’s disease virus in slaughter pigs from fattening pig herds. 

Journal of Veterinary Medicine, Serie B 46, 341-352. 

13. Maes, D.G., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., Verbeke, W. 

& Viaene, J. (1999b). Effect of vaccination against Mycoplasma hyopneumoniae in pig herds 

with an all-in/all-out production system. Vaccine 17, 1024-1034. 


(2000). Herd factors associated with the seroprevalences of four major respiratory pathogens in 

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15. Mateusen, B., Maes, D., Van Goubergen, M., Verdonck, M. & de Kruif, A. (2002). The 

effectiveness of strategic medication with lincomycin hydrochloride and/or vaccination against 

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16. Mycoplasma hyopneumoniae to control chronic respiratory disease in a swine herd. The 

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K.M. (1994). Persistence of passively acquired antibodies to Mycoplasma hyopneumoniae in a 

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K.M. (1995). Seroepidemiologic study of natural transmission of Mycoplasma hyopneumoniae 

in a swine herd. Preventive Veterinary Medicine 21, 323-337. 

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26. Stärk, K.D., Nicolet, J. & Frey, J. (1998). Detection of Mycoplasma hyopneumoniae by air 

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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE 

FIELD ISOLATES 


EVALUATION OF VIRULENCE OF MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES 

J. VICCA, T. STAKENBORG, D. MAES, P. BUTAYE, J. PEETERS, A. DE KRUIF & F. HAESEBROUCK 

VETERINARY MICROBIOLOGY 2003, 97, 177-190 

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2.1.2 EVALUATION OF VIRULENCE OF M. HYOPNEUMONIAE FIELD ISOLATES 

SUMMARY 

The course of enzootic pneumonia, caused by Mycoplasma hyopneumoniae, is 

strongly influenced by management and housing conditions. Other factors, including 

differences in virulence between M. hyopneumoniae isolates, may also be involved. 

The aim of this study was to evaluate the virulence of six M. hyopneumoniae field 

isolates and link it to genetic differences as determined by randomly-amplified 

polymorphic DNA (RAPD) analysis. 

Ninety, conventional M. hyopneumoniae-free piglets were inoculated 

intratracheally with the field isolates, a virulent reference strain or sterile culture 

medium. Animals were examined daily for the presence of disease signs and a 

respiratory disease score (RDS) was assessed per pig. Twenty-eight days post 

infection, pigs were euthanised, blood sampled and a lung lesion score was given. 

Lung samples were processed for histopathology, immunofluorescence testing for M. 

hyopneumoniae and isolation of M. hyopneumoniae. RAPD analysis was performed 

on all M. hyopneumoniae isolates. 

Significant differences between isolates were found for the RDS, lung lesion 

score, histopathology, immunofluorescence and serology. Based on the results of the 

different parameters, isolates were divided into 3 “virulence” groups: low, moderately 

and highly virulent isolates. Typically, a 5000 bp RAPD fragment was associated with 

the highly and moderately virulent isolates whereas it was absent in low virulent 

isolates. It was concluded that high variation in virulence exists between M. 

hyopneumoniae isolates isolated from different swine herds. Further studies are 

required to determine whether the 5000 bp fragment obtained in the RAPD analysis 

can be used as a virulence marker. 

- 87 -


INTRODUCTION 

Enzootic pneumonia in pigs, with M. hyopneumoniae as primary agent, is a 

chronic respiratory disease that causes major economic losses to the pig industry 

worldwide. The role of management and housing conditions in the development of 

enzootic pneumonia has been shown to be very important (Maes et al., 1999; Maes et 

al., 2000). Additional factors, different from housing and management conditions may 

also determine the infection pattern of M. hyopneumoniae and the clinical course of 

the infection (Vicca et al., 2002) since good conditions do not automatically result in 

a restricted course of enzootic pneumonia and poor conditions do not always result in 

severe disease. 

Several authors have demonstrated antigenic or genetic heterogeneity between 

M. hyopneumoniae isolates. Ro and Ross (1983) were able to demonstrate some 

diversity in antigenic structure; a different number of passages could cause a different 

pathogenicity in pigs (Zielinski and Ross, 1990). Frey et al. (1992) and Artiushin and 

Minion (1996) demonstrated genetic heterogeneity by means of restriction enzyme 

digestion and field inversion gel electrophoresis and RAPD, respectively. However, it 

is not known whether genetic heterogeneity correlates with differences in virulence. 

The aim of this study was to assess the virulence of M. hyopneumoniae 

isolates obtained from different herds (Vicca et al., 2002), using a well-standardised 

experimental infection model in conventional M. hyopneumoniae- and PRRSV-free 

pigs. RAPD analysis was used to determine the extent of genetic variability in the M. 

hyopneumoniae field isolates. 

- 88 -



M. hyopneumoniae isolates 

Six M. hyopneumoniae isolates obtained from the lungs of pigs from different 

Belgian farrow-to-finish herds were compared. Isolates 1, 2 and 3 were obtained from 

the lungs of pigs produced in herds that experienced a clinical course of enzootic 

pneumonia (chronic coughing in nursery and grow-finishing pigs, reduced production 

parameters in grow-finishing pigs). Isolates 4, 5 and 6 were obtained from herds with 

a subclinical course of enzootic pneumonia (no coughing or only some mild coughing 

in the last weeks of the grow-finishing period and the production parameters in the 

grow-finishing pigs reached target levels). All isolates originated from a different 

herd. Strain MP143 (kindly provided by Prof. Dr. N. Friis, SVS, Denmark) was 

isolated in Denmark from the lungs of a pig with mycoplasmal disease. 

Experimental design and animals 

Ninety, 3-week-old, cross-bred (Seghers hybrid, Belgium), castrated male pigs 

were obtained from a commercial herd that had no serological evidence of exposure 

to M. hyopneumoniae or porcine reproductive and respiratory syndrome virus 

(PRRSV). The source herd was a high health breeding herd in which repeated 

serological monitoring of sows and pigs of different age categories has been 

performed during the last 5 years. To detect antibodies against M. hyopneumoniae, the 

DAKO ® Mh ELISA (DAKO, Glostrup, Denmark) (Feld et al. 1992) was used. During 

at least five years, there was no clinical evidence of M. hyopneumoniae and no lung 

lesions typical for M. hyopneumoniae in slaughter pigs. 

The pigs were weaned at 21 days of age and moved to the animal facilities at 

the Faculty of Veterinary Medicine, Ghent University, Belgium. They were fed a 

commercial feed not containing antibiotics. Animals were weighed at 4 weeks of age, 

ranked according to weight and stratified into 3 groups. Within each weight group, 

pigs were randomly assigned to one of eight treatment groups. Each group of 10 

piglets was housed in rooms equipped with absolute filters (HEPA U15) to avoid 

spread of M. hyopneumoniae between groups. 

- 89 -


Immediately after weighing and allocation to the treatment groups, all pigs 

were anaesthetized with 0.22 ml/kg of a mixture of Xylm ® 2% (Intervet) and Zoletil 

100 ® (Virbac), ear tagged and inoculated intratracheally with 7 x 10 7 CCU of a M. 

hyopneumoniae isolate in 7 ml inoculum (groups inoculated with isolates 1-6 and 

isolate MP143) or with 7 ml sterile culture medium (negative control group). The 

group inoculated with strain MP143 served as positive control group. Because of the 

limited number of animal rooms equipped with absolute filters, the experiment was 

performed during 2 successive periods. A negative control group was used during 

both periods, resulting in a total of 20 negative control animals. The two negative 

control groups were considered as one group in the statistical analyses. The 

inoculation day was designated as day 0 post infection (0 DPI). 

The study was conducted after approval by the Ethical Committee for 

animal experiments of the Faculty of Veterinary Medicine, Ghent University. 

Clinical and performance parameters 

For 28 days following challenge, pigs were observed daily for 25 minutes, to 

evaluate body condition, appetite, presence of dyspnea and tachypnea and behavioral 

changes. In addition, a clinical respiratory disease score (RDS) was assessed daily 

from 0 to 28 days post infection (DPI) (Halbur et al., 1996). RDS scores could range 

from 0 to 6: 0 (no coughing), 1 (mild coughing after an encouraged move), 2 (mild 

coughing while leaving the pigs undisturbed), 3 (moderate coughing after encouraged 

move), 4 (moderate coughing while leaving the pigs undisturbed), 5 (severe coughing 

after encouraged move), 6 (severe coughing while leaving the pigs undisturbed). The 

number of coughing days was assessed as the total of days on which at least one pig 

was coughing. Rectal temperatures were measured on a daily basis from 0-10 DPI and 

every other day from 12-28 DPI. 

To calculate average daily weight gain (ADG), pigs were weighed every 

week, the first weighing took place at 0 DPI, the last weighing at 28 DPI (5 times in 

total). The amount of feed consumed during the trial (0-28 DPI) was measured to 

calculate the feed conversion ratio (FCR). 

- 90 -


Necropsy, lung lesions, immunofluorescence and bacteriological examinations 

All pigs were euthanised at 28 DPI by deep anesthesia with 0.3 ml/kg of a 

mixture of Xylm ® 2% (Intervet) and Zoletil 100 ® (Virbac) followed by 

exsanguination. The lungs were removed and the macroscopic pneumonic lesions 

were quantified using a lung lesion score diagram (Hannan et al., 1982). Total lung 

scores could vary between score 0 (no lesions) and a theoretical maximum of 35. A 

digital picture was taken from every lung for future reference. 

Lung tissue samples from every lobe were collected from the edge of a lung 

lesion, if present, and processed in a semi-quantitative immunofluorescence assay 

(score 0-3) to detect the presence of M. hyopneumoniae (Kobisch et al., 1978); 0: (no 

immunofluorescence), 1, 2 and 3 (limited, moderate and intense 

immunofluorescence). One score was given per lung lobe. 

Tissue samples from every lobe were also taken for histopathology. The tissue 

was fixed in 10% neutral buffered formalin and routinely processed and embedded in 

paraffin. The percentage of lung area occupied by air (percentage air) was examined 

using an automatic image analysis system (Optimas ® 6.5, Media Cybernetics, Silver 

Spring, USA), measuring the percentage of area occupied by air in the microscopic 

field (multiplication 400X). This percentage is inversely proportionate to the number 

of infiltrated cells, the amount of atelectasis, intrabronchiolar-intrabronchial exudate, 

intra-alveolar exudate and/or edema and proliferation of type II cells. An average 

percentage per inoculation group was calculated. This average was based on 700 

measurements: 10 microscopic fields per lung lobe, 7 lung lobes per lung and 10 pigs 

per inoculation group. The measurements were expressed as a percentage that could 

range from 0% through 100%. 

Using light microscopy, the severity of peribronchiolar and perivascular 

lymphohistiocytic infiltration and nodule formation consistent with M. 

hyopneumoniae induced pneumonia lesions were scored. The following classes were 

used: 0 (no cellular infiltrates), 1 (limited cellular infiltrates (macrophages and 

lymphocytes) around bronchioles, with airways and alveolar spaces free of cellular 

exudates), 2 (light to moderate infiltrates with mild diffuse cellular exudates into 

airways), 3, 4 and 5 (mild, moderate and severe lesions characteristic of bronchointerstitial 

pneumonia, centered around bronchioles but extending to the interstitium, 

- 91 -


with lymphofollicular infiltration and mixed inflammatory cell exudates) (Morris et 

al., 1995). An average score per inoculation group was calculated, based on 700 

measurements: 10 measurements per lung lobe, 7 lung lobes per lung and 10 pigs per 

inoculation group. 

For each inoculation group, pooled samples of 20 percent (w/v) suspensions of 

lungs were made, inoculated on Columbia agar supplemented with 5 percent sheep 

blood (blood agar) and incubated at 37°C for 48 hours to check the presence of 

secondary bacteriological organisms in the lungs. The plates were cross-streaked with 

Staphylococcus aureus for support of Actinobacillus pleuropneumoniae growth. 

M. hyopneumoniae isolation 

For each inoculation group, pooled samples of 20 percent (w/v) suspensions of 

lungs in phosphate buffered saline solution were used for isolation of M. 

hyopneumoniae. Isolation and cultivation of M. hyopneumoniae was optimised using 

earlier reports (Friis 1971a,b; 1975; 1979). Briefly, lung suspensions were inoculated 

into selective Friis medium. A series of subcultures in broth media were performed, 

followed by growing M. hyopneumoniae colonies on solid media. The mycoplasma 

species (M. hyopneumoniae, M. hyorhinis or M. flocculare) growing in broth media or 

on solid media were identified using a species specific PCR (Mattsson et al., 1995; 

Caron et al., 2000). For identifying M. flocculare species following primers were used 

(Stakenborg et al., 2005): 

MYCO REV 

AGAGGCATGATGATTTGACGTC 

M FLOC 741FOR TTAGGTAGGGAATGATCTAATC 

Single M. hyopneumoniae colonies were picked up from plate and transferred 

to broth medium. 

Serology for M. hyopneumoniae 

A blood sample of every pig was taken at 0 and 28 DPI to detect antibodies 

against M. hyopneumoniae, using the DAKO ® Mh ELISA (DAKO, Glostrup, 

Denmark) (Feld et al., 1992). Sera with optical density (OD)-values


OD-values were considered doubtful and classified as negative in the statistical 

analysis. 

Randomly amplified polymorphic DNA (RAPD) 

RAPD analysis was performed on all M. hyopneumoniae isolates. The 

protocol used was mainly based on the article of Artiushin and Minion (1996). 

Briefly, 45 amplification cycles (1 min. at 95°C; 1 min. at 36°C; and 2 min. at 72°C) 

were run on a GeneAmp 2400 Thermal Cycler (Perkin Elmer, USA). The DNA of the 

M. hyopneumoniae isolates used was firstly purified using a DNA easy Kit 

(Westburg, The Netherlands) to increase reproducibility. The 30 µl-reaction mixture 

was optimised containing 30 ng of purified genomic DNA, 2 mM MgCl 2 , 20 pmol of 

OPA-3 primer, 120 uM dNTPs, 1x Amplitaq Buffer, 1x Stoffel Buffer, 1.5 U 

AmpliTaq DNA polymerase and 0.75 U Amplitaq DNA polymerase Stoffel Fragment 

(Perkin Elmer). The amplified fragments were seperated using standard gelelectrophoresis 

technique (90V, for 2 hours in TBE buffer). Smartladder (Eurogentec, 

Belgium) was used as size-marker. The bands were visualized and analysed with 

Bionumerics software (Applied Maths, Belgium). 

Statistical analyses 

Analysis of variance (ANOVA) was used to compare the results of the 

different inoculation groups and to link the presence or absence of a 5000bp fragment 

determined in RAPD to the results of the different M. hyopneumoniae inoculation 

groups. A Bonferroni correction was used to adjust the observed significance level for 

the fact that multiple comparisons were made. To estimate correlations between 

parameters, a bivariate analysis with Spearman’s correlation coefficient was used. 

The prevalence of pigs positive for the different parameters was compared 

between inoculation groups by logistic regression. Statistical results were considered 

to be significant when P-values were lower than 0.05 (two-tailed). Data were analyzed 

using the statistical package SPSS 10.0 for Windows (SPSS 10, SPSS Inc. Illinois 

60611 USA 1999). 

- 93 -


RESULTS 

Results are summarized in Table 1 

Clinical evaluation and performance parameters 

One of the pigs inoculated with isolate 6 died at 17 DPI because of a rupture 

of the urinary bladder. The average rectal temperatures were not significantly 

different between inoculation groups (individual temperatures ranged from 38.2-41.2 

°C). Rectal temperatures slowly decreased towards the end of the study in all groups. 

Coughing was present in all groups inoculated with M. hyopneumoniae but was rarely 

observed in the negative control group (Figure 1). Coughing started at approximately 

7 DPI and increased steadily towards the end of the study. The incubation period 

varied according to the isolate used. The average RDS was significantly higher 

(P


3 

2.5 

2 

Average RDS 

1.5 

1 

0.5 

0 

-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 

DPI 

Negative Control Positive Control 1 

2 3 4 

5 6 

Figure 1: Average respiratory disease Score (RDS) from -2 DPI until 28 days post 

inoculation (DPI) for pigs inoculated with sterile culture medium (negative control), 

strain MP143 (positive control) or Belgian M. hyopneumoniae field isolates (1-6). 

Macroscopic lung lesions and bacteriological examinations 

The lung lesion scores of pigs inoculated with strain MP143 or isolate 1, were 

significantly higher compared to the negative control group (P


Potential secondary pathogens as Pasteurella multocida, Bordetella 

bronchiseptica and Streptococcus suis were found in lungs of pigs in all inoculation 

groups, including the negative control group. The prevalences of pig lungs infected 

with potential secondary pathogens were 30%, 10%, 30%, 20%, 50%, 50%, 50% and 

20% for the negative control group, positive control group and groups 1 to 6, 

respectively. Actinobacillus pleuropneumoniae was not isolated from any of the pig 

lungs. The proportions of pigs infected with secondary pathogens were not 

significantly different between the inoculation groups. 

Microscopic lung lesions 

Compared to the negative control group and groups inoculated with isolates 2, 

4 and 6, a significantly (P


Immunofluorescence (IF) and isolation 

M. hyopneumoniae organisms were not detected in the negative control group. 

The positive control group and the group inoculated with isolate 1 showed a 

significantly (P


the group inoculated with isolate 2 (P


- 99 -


Correlation of parameters 

The parameters used to compare the virulence of the different M. 

hyopneumoniae isolates correlated well (r > 0.56) (Table 2). Percentage air and 

serology (OD-values) were negatively correlated with the other parameters (RDS, 

number of coughing days, lung lesions, lymphohistiocyte infiltration and 

immunofluorescence). 

Table 2: Spearman’s correlation coefficients of parameters used to evaluate virulence 

of the M. hyopneumoniae field isolates 

Respiratory 

Disease 

Score 

Number 

of 

coughing 

days 

Lymphohistiocytic 

infiltration 

Percentage 

air 


Lung 

lesions 

Respiratory 

1.000 0.991 0.662 - 0.643 0.755 0.804 - 0.736 

Disease Score 

Number of 

1.000 0.644 - 0.636 0.769 0.811 - 0.735 

coughing days 

Lymphohistiocytic 

1.000 - 0.604 0.705 0.670 - 0.704 

infiltration 

Percentage air 1.000 - 0.604 - 0.604 0.564 

Serology 

(ODvalues) 


1.000 0.893 - 0.780 

Lung lesions 1.000 - 0.815 

Serology 

(OD-values) 

1.000 

All correlations were significant at the 0.01 level 

- 100 -


DISCUSSION 

This study proved the hypothesis that differences in virulence between 

circulating M. hyopneumoniae field isolates exist. This hypothesis was based on 

observed different clinical courses of the disease in the herds selected for isolation of 

the isolates. Although the importance of management and housing conditions has 

been shown several times (Clark et al., 1991; Ross, 1999) and should never be 

underestimated, earlier field experiments performed at our department (Vicca et al., 

2002) showed that additional factors different from housing and management 

conditions may determine the infection pattern of M. hyopneumoniae and the clinical 

course of the infection. 

It has been shown that a different number of in vitro passages can result in a 

different pathogenicity of M. hyopneumoniae isolates in pigs (Whittlestone, 1979; 

Zielinsky and Ross, 1990). In the present studies, the number of passages used to 

obtain pure cultures of isolates 1, 3, 4, 5 and 6 varied between 8 and 10, 

demonstrating that differences in virulence between these isolates were not due to 

different numbers of in vitro passages. Isolate 2 was passed 20 times in vitro. It 

cannot be excluded that the higher number of passages played a role in decreased 

virulence of this isolate. 

Performance parameters such as ADG and FCR were of little use in this study 

to evaluate differences in virulence. Although these parameters are very important to 

evaluate the economical results of a swine herd, the number of animals used and the 

duration of the study period were probably too limited to observe an effect of the M. 

hyopneumoniae inoculation on these performance parameters. 

The parameters RDS, lung lesion score, infiltration of lymphohistiocytes, 

percentage of air left in the lung, immunofluorescence and serology appeared to be 

important to evaluate the virulence of isolates during this study. A good correlation 

between the above-mentioned parameters was demonstrated (Table 2). Kobisch et al. 

(1993) and Morris et al. (1995) already found a positive correlation between coughing 

and lung lesion scores. In an earlier study from Zielinsky and Ross (1990), no 

consistent relation between microscopic lesions and the extent of macroscopic lesions 

was found; this is in contradiction with our results. Zielinsky and Ross (1990) 

suggested that the extent of macroscopic lesions was rather influenced by natural 

- 101 -


susceptibility (attributable to genetic components) of the pigs than by the action of the 

organism itself. All pigs used in our study were born in the same herd, a breeding herd 

that strives for genetic homogeneity. The genetic variability between pigs could not 

have been sufficiently large to be the only explanation for the observed variability in 

extent of macroscopic lesions between inoculation groups. 

Based on the outcomes of the clinical and pathological parameters, the isolates 

could roughly be divided into 3 groups: highly virulent isolates (strain MP143 and 

isolate 1), moderately virulent isolates (3 and 5) and low virulent isolates (2, 4 and 6). 

For all parameters, highly virulent isolates differed significantly from the negative 

control group. Low virulent isolates did not show significant differences from the 

negative control group, but did show significant differences from the highly virulent 

isolates for the parameters: RDS, lung lesion score, percentage air, lymphohistiocytic 

infiltration (except isolate 2) and IF (except isolate 4). The isolates 3 and 5 could 

statistically not be distinguished clearly from the other groups: the results of RDS and 

macroscopic lung lesions were more in accordance with the results of the low virulent 

isolates, the results from the microscopic lesions, IF and serology were more in 

accordance with the results of the highly virulent isolates. Therefore, these isolates 

were considered as moderately virulent. Since microscopic lesions due to M. 

hyopneumoniae infection are evident at an earlier time than macroscopic lesions, a 

longer study period (> 28 days) could have resulted in a more severe RDS and lung 

lesion score. 

The intensity of immunofluorescence was clearly higher in the lungs of pigs 

inoculated with the highly virulent isolates. This might indicate that the highly 

virulent isolates had a higher capacity to colonize or attach to the ciliated epithelium 

or were able to multiply faster resulting in more organisms causing an earlier and 

more severe coughing, more severe macroscopic and microscopic lesions and more 

intense immunofluorescence. A difference in capability to attach to cilia between M. 

hyopneumoniae isolates has been demonstrated by Minion et al. (2000) and was 

correlated with the number of repeat regions in RR1 of the P97 protein of M. 

hyopneumoniae. A minimum of eight repeats was necessary to make the M. 

hyopneumoniae organisms able to attach to the cilia. Research on the P97 protein of 

the different M. hyopneumoniae isolates used in this study could provide important 

information about the attachment capacities of the isolates and could partly explain 

the differences in virulence between isolates observed in this study. 

- 102 -


Piglets inoculated with a highly virulent isolate had a higher number of piglets 

seroconverted at 28 DPI compared to piglets inoculated with a low virulent isolate. 

An earlier seroconversion of 3 weeks was also seen in herds experiencing a clinical 

course of enzootic pneumonia compared to herds subclinically infected with M. 

hyopneumoniae (Vicca et al., 2002). The reason for this finding is not clear but it 

might be because of a higher immunogenicity or a faster multiplication of the highly 

virulent isolates. A difference in glycosylation of immunogenic proteins between M. 

hyopneumoniae isolates was described by Lin (2001), but no relation to virulence of 

the isolates was made. Differences in immunogeneity between different M. 

hyopneumoniae isolates could be of special interest for future vaccine development. 

A 5000 bp RAPD band was only present in highly and moderately virulent 

isolates and not in low virulent isolates. Further research is required to determine 

whether this band can be used as a virulence marker for M. hyopneumoniae. 

CONCLUSION 

In the present study, variation in virulence between different field isolates of 

M. hyopneumoniae was demonstrated in an experimental infection model. Further 

studies are required to determine the usefulness of the 5000 bp RAPD band to predict 

virulence of M. hyopneumoniae isolates. 


This research was financially supported by grant S-5940 (section II) of the 

Belgian Ministry of Small Enterprises, Trader and Agriculture, Research Foundation 

(Brussels). I am grateful to all the people who assisted me with the practical aspects of 

this study. 

- 103 -


REFERENCES 

1. Artiushin, S. & Minion, F.C. (1996). Arbitrarily primed PCR analysis of Mycoplasma 


Systemic Bacteriology 46, 324-328. 

2. Caron, J., Ouardani, M. & Dea, S. (2000). Diagnosis and differentiation of Mycoplasma 

hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 

and p46 genes. Journal of Clinical Microbiology 38, 1390-1396. 

3. Clark, L., Armstrong, C., Knox, K. & Mayrose, V. (1991). The effect of all-in/all-out 

management on pigs from a herd with enzootic pneumonia. Veterinary Medicine 86, 946-951. 

4. Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F. & Meyling, A. (1992). A monoclonal blocking 


35-46. 

5. Frey, J., Haldimann, A. & Nicolet, J. (1992). Chromosomal heterogeneity of various 


275-280. 

6. Friis, N.F. (1971ª). A selective medium for Mycoplasma suipneumoniae. Acta Veterinaria 


7. Friis, N.F. (1971b). Mycoplasmas cultivated from the respiratory tract of Danish pigs. Acta 

Veterinaria Scandinavia 12, 69-79. 

8. Friis, N.F. (1975). Some recommendations concerning primary isolation of Mycoplasma 

suipneumoniae and Mycoplasma flocculare. Nordisk Veterinaer Medicin 27, 337-339. 

9. Friis, N.F. (1979). Selective isolation of slowly growing acidifying mycoplasmas from swine 

and cattle. Acta Veterinaria Scandinavia 20, 607-609. 

10. Halbur, P.G., Paul, P.S., Meng, X.J., Lum, M.A., Andrews, J.J. & Rathje, J.A. (1996). 

Comparative pathogenicity of nine US Porcine Reproductive and Respiratory Syndrome Virus 

(PRRSV) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model. Journal of 

Veterinary Diagnostic Investigation 8, 11-20. 

11. Hannan, P.C., Bhogal, B.S. & Fish, J.P. (1982). Tylosin tartrate and tiamutilin effects on 



12. Kobisch, M., Tillon, J.P., Vannier, Ph., Magueur, S. & Morvan, P. (1978). Pneumonie 

enzootique à Mycoplasma suipneumoniae chez le porc: diagnostic rapide et recherche 

d’anticorps. Recueil de Medecine Veterinaire 154, 847-852. 

13. Kobisch, M., Blanchard, B. & Le Potier, M.F. (1993). Mycoplasma hyopneumoniae infection 

in pigs: duration of the disease and resistance to reinfection. Veterinary Research 24, 67-77. 

14. Lin, B. (2001). Intraspecies differentiation of Mycoplasma hyopneumoniae field strains isolated 

in the United States. American Association of Swine Veterinarians, 225-235. 

15. Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B. & de Kruif, A. 

(1999). Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine 

Influenza viruses and Aujeszky’s disease virus in slaughter pigs from fattening pig herds. 

Journal of Veterinary Medicine, Series B 46, 341-352. 

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(2000). Herd factors associated with the seroprevalences of four major respiratory pathogens in 


17. Mattsson, J.G., Bergström, K., Wallgren, P. & Johansson, K.E. (1995). Detection of 

Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16s RNA 

gene. Journal of Clinical Microbiology 33, 893-897. 

18. Minion, F.C., Adams, C. & Hsu, T. (2000). R1 region of P97 mediates adherence of 


19. Morris, C.R., Gardner, I.A., Hietala, S.K. & Carpenter, T.E. (1995). Enzootic pneumonia: 

comparison of cough and lung lesions as predictors of weight gain in swine. Canadian Journal of 


20. Ro, L.H. & Ross, R.F. (1983). Comparison of Mycoplasma hyopneumoniae strains by serologic 

methods. American Journal of Veterinary Research 11, 2087-2094. 

21. Ross, R.F. (1999). Mycoplasmal Diseases. In: Leman A.D., Straw B.E., Mengeling W.L., 

Allaire S.D., Taylor D.J (Eds.), Diseases of Swine, 8th Edition, Iowa State University Press, 

Ames, pp. 537-551. 

22. Stakenborg, T., Vicca, J., Butaye, P., Imberechts, H., Peeters, J., de Kruif, A., 

Haesebrouck, F., Maes, D. (2005). A multiplex PCR to identify mycoplasmas present in broth 

cultures. Veterinary Research Communications, In press. 

23. Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F. & de Kruif, A. (2002). 

Patterns of Mycoplasma hyopneumoniae infections in Belgian farrow-to-finish pig herds with 

diverging disease-course. Journal of Veterinary Medicine, Series B 49, 349-353. 

24. Whittlestone, P. (1979). Porcine mycoplasmas. In: Tully, J.G. and Whitcomb, R.F. (Ed.), The 

Mycoplasmas II. Human and Animal Mycoplasmas, Academic press, Inc., New York, pp. 133- 

176. 

25. Zielinsky, G. & Ross, R. (1990). Effect of growth in cell cultures and strain on virulence of 

Mycoplasma hyopneumoniae for swine. American Journal of Veterinary Research 51, 344-348. 

- 105 -

2.2 SUSCEPTIBILITY OF M. HYOPNEUMONIAE TO 

ANTIMICROBIAL AGENTS 

- 107 -

2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD 

ISOLATES 


IN VITRO SUSCEPTIBILITIES OF MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES 

J. VICCA, T. STAKENBORG, D. MAES, P. BUTAYE, J. PEETERS, A. DE KRUIF & F. HAESEBROUCK 

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 2004, 48, 4470-4472 

- 109 -

2.2.1 IN VITRO SUSCEPTIBILITY OF M. HYOPNEUMONIAE FIELD ISOLATES 

SUMMARY 

Mycoplasma hyopneumoniae causes enzootic pneumonia, a chronic 

respiratory disease in pigs. To control the infection, preventive and curative use of 

antibiotics in feed or water is a common practice. Unfortunately, susceptibility of M. 

hyopneumoniae to different antimicrobials has rarely been determined. In this study, a 

broth microdilution technique was used to determine the in vitro susceptibilities of 21 

M. hyopneumoniae field isolates. Acquired resistance to spectinomycin, 

oxytetracycline, doxycycline, gentamicin, florfenicol and tiamulin was not observed. 

One isolate showed acquired resistance to lincomycin, tilmicosin and tylosin. This 

isolate was susceptible to all other antibiotics tested. The MIC-values of flumequine 

were > 16 µg/ml for 5 isolates, while the MIC 50 -value was 2 µg/ml. For these 5 

isolates, the MIC-values for enrofloxacin were ≥ 0.5 µg/ml, the MIC 50 being 0.06 

µg/ml. As far as we know, this is the first report of acquired resistance against 

macrolides, lincosamides and fluoroquinolones in M. hyopneumoniae field isolates. 

- 111 -


INTRODUCTION 

Mycoplasma hyopneumoniae causes enzootic pneumonia, a chronic 

respiratory disease in pigs resulting in considerable economic losses. Although 

appropriate vaccines are available to reduce the consequences of infection, in feed or 

water medication with antimicrobials is still a common practice to treat or control the 

disease. The use of antimicrobials, however, results in the selection of resistant 

bacteria. Antimicrobial susceptibility of M. hyopneumoniae field isolates has rarely 

been determined and limited numbers of isolates have been considered in these 

studies (Hannan et al., 1989; Ter Laak et al., 1991; Tanner et al., 1993; Friis and 

Szancer, 1994; Hannan et al., 1997a; Hannan et al., 1997b). This is mainly due to the 

fact that M. hyopneumoniae is a very fastidious and slowly growing micro-organism 

which makes it difficult to obtain large numbers of field isolates. In addition, 

susceptibility testing is more difficult for these organisms compared to other bacteria 

as there is no standardized procedure. Recently, guidelines and recommendations for 

MIC testing against veterinary mycoplasma species have been proposed on behalf of 

the International Research Programme on Comparative Mycoplasmology (IRPCM) 

(Hannan, 2000). 

In the present study, antimicrobial susceptibility of M. hyopneumoniae 

isolates, recently obtained from swine in the field, was determined using these 

guidelines and recommendations. Also, antibiotic use on the herds where the isolates 

where obtained was monitored in order to try to find a possible link between antibiotic 

use and MIC results. 



Twenty-one M. hyopneumoniae field isolates, obtained between 2000 and 

2002 from the lungs of pigs from 21 different farrow-to-finish pig herds in Belgium, 

were used for MIC determination (Vicca et al., 2002; Vicca et al., 2003). All isolates 

were obtained from the lungs of slaughter pigs, except isolates 1, 4, 5 and 6 which 

were obtained from the lungs of 9-12 week old euthanized pigs which did not present 

- 112 -


respiratory tract disease signs. The antibiotics used on the different herds in suckling 

piglets (0 - 4 weeks of age), nursery piglets (4 - 10 weeks of age) and growth - 

finishing pigs (10 weeks until slaughter) during the year before the M. hyopneumoniae 

isolates were obtained, are mentioned in Table 1. 

Isolation and cultivation of M. hyopneumoniae was optimized using earlier 

reports (Friis 1971a,b; 1975; 1979). Briefly, lung suspensions were inoculated into 

selective Friis medium. A series of subcultures in selective and non-selective Friis 

medium were carried out, followed by growing M. hyopneumoniae colonies on solid 

medium (agar mixed with non-selective Friis medium). M. hyopneumoniae was 

identified using a species specific PCR (Mattsson et al., 1995; Caron et al., 2000). 

Single M. hyopneumoniae colonies were picked up from plate and transferred 

to non-selective Friis medium. Isolates were stored at -70°C until used. 

The M. hyopneumoniae type strain ATCC 25634 (J-strain) was used as control strain. 

- 113 -


Table 1: The antibiotics used during 1 year before isolation of M. hyopneumoniae on 

the herd 

Herd Isolate Antibiotics b used in 

Suckling piglets Nursery piglets Grow-finishing pigs 

A 1 ENRO 1, a ENRO 1 OXY 1 

B 2 DANO 1 COL 5 none 

C 4* ENRO 1 COL 5 TYL 4 or PEN 1 

D 5 AMK 2 AMK+COL 5 OXY 3 or DOX 3 

E 6 AMK 2 or PEN 2 TMP+SULF 3 TYL 3 or DOX 3 

F 7 ENRO 1 COL 4 , APR 4 , AMK 4 , None 

OXY 4 , ENRO 1 

G 8* PEN 2 , ENRO 1 , NEO 1 LIN 1 , OXY 6 , TYL 6 LIN 1 , OXY 6 , TYL 6 

H 9 AMK 2 LIN 1 , OXY 6 LIN 1 , OXY 6 

I 10 none COL 3 none 

J 11 none AMK 3 none 

K 12 none COL 5 none 

L 13 

AMK 1 , ENRO 1 , PEN 2 AMK 1 , APR 3 , COL 3 , 

DOX 4 , ENRO 1 , 

CEF 1 , TYL 1 

TMP+SULF 3 , 

M 14* ENRO1, PEN+STR 1 , AMK 5 , COL 5 , ENRO 2 , OXY 5 

CEF 1 , AMK 1 CEF 1 , PEN+STR 1 

N 15 none none TMP+SULF 6 

O 16 TMP+SULF 1 , AMK 1 OXY 1 OXY 1 

P 17* AMK 1 , ENRO 1 AMK 5 , AMK 1 DOX 5 , FFN 1 

Q 18 CEF 1 , ENRO 1 COL 5 , TMP+SULF 4 none 

R 19** none none LIN 1 

S 20* ENRO 1 ENRO 1 , FLUM 3 ENRO 1 , PEN 1 , OXY 1 

T 21 none none DOX 4 

U 23 COL 1 , AMK 2 COL 5 TYL+DOX 5 

a : 

1 : injectable (some pigs injected in case of disease); 2 : injectable (all pigs injected, routine ); 3 : 

in feed medication (routine); 4 : in feed medication (in case of disease); 5 : in water medication 

(routine); 6 : in water medication (in case of disease) 

b 

: AMK, amoxicillin; APR, apramycin; CEF, cefquinome; COL, colistin; DANO, danofloxacin; 

DOX, doxycycline; ENRO, enrofloxacin; FFN, florfenicol; FLUM, flumequine; LIN, 

lincomycin; OXY, oxytetracycline; PEN, penicillin; PEN+STR, penicillin and streptomycin; 

TMP+SULF, trimethoprim and sulfamides; TYL, tylosin 

*: High MIC values for flumequine and enrofloxacin 

**: High MIC values for tylosin, tilmicosin and lincomycin 

Minimal inhibitory concentration (MIC) determination 

Ninety-six well, round-bottom microtitre plates (Sensititre Ltd., Imberhorne 

Lane, East Grinstead, Sussex, England) containing stabilized freeze-dried lincomycin 

(0.06 - 8 µg/ml), lincomycin / spectinomycin 1:2 ratio (0.06/0.12 - 8/16 µg/ml), 

spectinomycin (0.12 - 16 µg/ml), oxytetracycline (0.015 - 2 µg/ml), doxycycline 

(0.015 - 2 µg/ml), enrofloxacin (0.008 - 1 µg/ml), flumequine (0.12 - 16 µg/ml), 

gentamicin (0.12 - 16 µg/ml), florfenicol (0.12 - 16 µg/ml), tiamulin (0.015 - 1 

- 114 -


µg/ml), tilmicosin (0.25 - 16 µg/ml) and tylosin tartrate (0.015 - 1 µg/ml) were used. 

Three wells on each plate were left antimicrobial-free as a positive growth control. 

M. hyopneumoniae isolates were removed from cryostorage (-70°C), allowed 

to thaw at room temperature and incubated during 1-2 hours in non-selective Friis 

medium at 36 ± 1 °C. Isolates were vortexed and diluted in non-selective Friis 

medium until the number of organisms reached 10 4 CCU/ml. Fifty µl of the diluted 

culture was transferred into each well of the Sensititre® plate. The J-strain was tested 

3 times in order to estimate reproducibility of the procedure. After inoculation, the 

microtiter plates were sealed using an adhesive foil and incubated at 36 ± 1 °C for 14 

days. Plates were observed daily. Growth of M. hyopneumoniae organisms was 

observed when the colour of the medium changed from red to yellow (phenol red 

indicator), corresponding with a decrease in pH from 7.4 to 6.8 by glucose 

fermentation. The initial and final MICs were recorded. The initial MIC was defined 

as the lowest antibiotic concentration to show no change in colour when the colour of 

the growth control turned yellow. The final MIC was defined as the lowest antibiotic 

concentration to show no change in colour at 14 days after inoculation (Tanner and 

Wu, 1992). 

RESULTS 

In Table 2, the initial and final MIC 50 , MIC 90 and MIC-range are presented 

for the 21 M. hyopneumoniae field isolates. Only for oxytetracycline, doxycycline and 

gentamicin the initial and final MIC 50 differed from each other for more than one 

dilution. No clear differences were detected between initial and final MIC 90 values. 

The MIC-values for the 3 replicates of the J-strain are also given in Table 2. It can be 

seen that these values were equal or differed from each other for only one doubling 

dilution, indicating a good reproducibility of the test. The initial MIC-values for the J- 

strain were in agreement with the values reported in earlier studies (Hannan et al., 

1989; Ter Laak et al., 1991; Tanner et al., 1993; Hannan et al., 1997a,b). 

In Table 3, frequency distributions of final MIC values of the different 

antibiotics for the 21 field isolates are presented. 

The MICs of oxytetracycline, doxycycline, tiamulin, spectinomycin, 

gentamicin, florfenicol and the lincomycin - spectinomycin combination did not show 

any indication of a bimodal frequency distribution range (Butaye et al., 2003). For 

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one isolate, obtained on farm R, the MICs of tylosin, tilmicosin and lincomycin were 

clearly higher than for the other isolates. Isolates number 4, 8, 14, 17 and 20 had a 

MIC value of > 16 µg/ml for flumequine while the final MIC 50 equals only 2 µg/ml, 

corresponding to the median of the first distribution. For the same isolates the MIC 

value of enrofloxacine was ≥ 0.5 µg/ml while the MIC 50 was 0.06 µg/ml. 

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Table 2: Initial and final MIC 50 , MIC 90 en MIC-range (µg/ml) of antimicrobials 

against Belgian M. hyopneumoniae field isolates. The MIC for the reference strain (Jstrain) 

was determined 3 times and the results are included is this table. 

Antimicrobial 1 Reading Field isolates (n=21) Reference strain: 

J-strain 

MIC 50 MIC 90 MIC-Range MIC-range 

LIN Initial ≤ 0.06 ≤ 0.06 ≤ 0.06 - > 8 8 0.25 

LIN/SPT Initial ≤ 0.06/0.12 ≤ 

0.06/0.12 

≤ 0.06/0.12 - 

0.25/0.5 

Final ≤ 0.06/0.12 0.12/0.25 ≤ 0.06/0.12 - 

0.25/0.5 

2 1 

DOX Initial 0.12 0.5 0.03 - 1 0.06 - 0.12 

Final 0.5 1 0.12 - 2 0.5 - 1 

ENRO Initial 0.03 0.5 0.015 - > 1 0.015 - 0.03 

Final 0.06 0.5 0.03 - > 1 0.06 

FLUM Initial 1 > 16 0.25 - > 16 0.5 - 1 

Final 2 > 16 0.5 - > 16 2 

GEN Initial ≤ 0.12 0.5 ≤ 0.12 - 1 0.25 - 0.5 

Final 0.5 1 ≤ 0.12 - 1 0.5 - 1 

FFN Initial ≤ 0.12 0.25 ≤ 0.12 - 0.5 0.25 

Final 0.25 0.5 ≤ 0.12 - 1 1 

TIA Initial ≤ 0.015 0.12 ≤ 0.015 - 0.12 0.03 

Final 0.03 0.12 ≤ 0.015 - 0.12 0.06 

TIL Initial 0.25 0.5 ≤ 0.25 - > 16 0.25 

Final 0.5 0.5 ≤ 0.25 - > 16 0.5 

TYL Initial 0.03 0.06 ≤ 0.015 - > 1 1 0.12 

1 : LIN, lincomycin; SPT, spectinomycin; OXY, oxytetracycline; DOX, doxycycline; ENRO, 

enrofloxacin; FLUM, flumequine; GEN, gentamicin; FFN, florfenicol; TIA, tiamulin; TIL, 

tilmicosin; TYL, tylosin 

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Table 3: Frequency distribution of final minimal inhibitory concentrations (MICs) of 

12 antibiotics on 21 Belgian M. hyopneumoniae field strains. 

Antimicrobial 1 

Number of stains with MIC (µg/ml) of 

0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 

Lincomycin 16 4 3 1** (>)* 

Lincomycin/ 

17 4 3 

Spectinomycin 

Spectinomycin 3 (≤ ) 2 8 11 

Oxytetracycline 5 2 5 5 6, 1 (>) 

Doxycycline 5 3 5 9 2 

Enrofloxacin 5 13 1 4 1 (>) 

Flumequine 3 3 11 2 5 (>) 

Gentamicin 4 (≤ ) 4 10 6 

Florfenicol 7 (≤ ) 8 6 3 

Tiamulin 4 10 7 3 

Tilmicosin 10 (≤ ) 13 1 (>) 

Tylosin 2 5 6 10 1 (>) 

*: > : higher than MIC indicated 

≤: equal or lower than MIC indicated 

**: isolates considered to have acquired resistance are underlined 

DISCUSSION 

In the present study a bimodal frequency distribution of MICs for the 

macrolides tylosin and tilmicosin as well as for the lincosamide antibiotic, lincomycin 

was seen. The MICs of these antibiotics were clearly higher for one isolate, indicating 

acquired resistance. The MIC of tylosin for this isolate was also higher than the 

breakpoint proposed by Hannan et al. (1997a). Macrolides and lincosamides are 

chemically distinct but they have a similar mode of action and, moreover, overlapping 

binding sides on the 23S rRNA of the 50S subunit of the bacterial ribosome. They act 

by blocking protein synthesis on assembled and functioning 50S ribosomal subunits 

(Weisblum, 1995; Butaye 2000). Acquired resistance against these antibiotics has not 

been described before in M. hyopneumoniae and the mechanism of resistance is 

unknown. In M. pneumoniae, acquired resistance against macrolides has been 

associated with an alteration in the drug target side by point mutations in the 23s 

rRNA gene (Lucier et al., 1995). 

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Acquired resistance to macrolides and lincosamides in mycoplasmas has not 

been reported often, most probably due to a limited amount of isolates tested. Only 2 

M. pneumoniae, 1 Ureaplasma spp. and 2 M. hominis resistant strains were isolated 

from humans (Bébéar and Bébéar, 2002). In animal mycoplasmas, acquired resistance 

to tylosin has been described for M. gallisepticum (Levisohn, 1981), M. hyosynoviae 

(Kobayashi et al., 1996b; Aarestrup et al. 1998), M. hyorhinis (Kobayashi et al., 

1996a,b) and M. bovis (Thomas et al., 2003). No co-resistance to lincomycin was 

observed for the tylosin resistant M. hyosynoviae isolates. Lincomycin was not 

evaluated by Levisohn (1981), and although tylosin and lincomycin resistant M. bovis 

isolates were found by Thomas et al. (2003), the existence of co-resistance was not 

reported. Ter Laak et al. (1993) found M. bovis isolates that were resistant to 

lincomycin, but they were susceptible to tylosin. The use of lincomycin in growfinishing 

pigs on the herd where our macrolide-lincosamide resistant M. 

hyopneumoniae isolate was obtained may have contributed to selection of antibiotic 

resistance. 

The MICs of flumequine and enrofloxacin also showed an extended frequency 

distribution range with a tendency towards bimodality. For 5 of the 21 isolates the 

MIC of flumequine was >16 µg/ml, which is higher than the breakpoint of ≥ 16 µg/ml 

proposed by Hannan et al. (1997a). For these isolates, the MIC of enrofloxacin was ≥ 

0.5 µg/ml while the MIC 50 was 0.06 µg/ml. This rather high frequency of acquired 

resistance against fluoroquinolones is unusual. Although fluoroquinolone resistant M. 

hominis isolates have been reported (Bébéar et al., 1999), resistance against these 

antibiotics has been rarely reported in human associated mycoplasmas. Hannan et al. 

(1997a) described acquired resistance against flumequine in avian, porcine (but not M. 

hyopneumoniae), bovine, ovine and caprine mycoplasmas and Thomas et al. (2003) 

isolated enrofloxacin resistant strains from bovines. It has also been shown that in 

vitro passages of mycoplasmas in the presence of enrofloxacin results in the 

development of fluoroquinolone resistance (Gautier-Bouchardon et al., 2002; 

Reinhardt et al., 2002; Bébéar et al., 1997). A possible explanation for the high 

prevalence of fluoroquinolone resistance in the present study might be the frequent 

use of enrofloxacin to treat E. coli diarrhea in neonatal and recently weaned piglets. In 

all the herds with a fluoroquinolone resistant M. hyopneumoniae isolate, these 

antibiotics were used during the suckling period. On only 5 herds with a fully 

susceptible M. hyopneumoniae isolate, fluoroquinolones were sporadically used to 

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treat suckling piglets. The MIC of enrofloxacine of M. hyopneumoniae isolate 20 was 

>1. On the originating herd fluoroquinolones were used as well in suckling, nursery 

and grow-finishing pigs. 

The MICs of oxytetracycline and doxycycline did not show a clear bimodal 

frequency distribution range and the MICs of oxytetracycline were lower than the 

breakpoint proposed by Hannan et al. (1997a), indicating no acquired resistance 

against these antibiotics although 62 % of the herds selected for this study used 

tetracycline antibiotics to treat nursery and grow-finishing pigs. M. hyopneumoniae 

isolates with decreased oxytetracycline susceptibility have been isolated in Japan 

(Inamoto et al., 1994). Acquired resistance against chlortetracycline has been 

described by Yamamoto and Koshimizu (1984), Inamoto et al. (1994) and Etheridge 

et al. (1979). For tetracycline resistance we used the antibiotics oxytetracycline and 

doxycycline since stability of chlortetracycline is rather poor (Ray and Newton, 1991) 

and due to the long incubation period necessary, one cannot be sure of the activity 

after this prolonged period of incubation. 

CONCLUSION 

This study is the first description of acquired resistance in M. hyopneumoniae 

field isolates to macrolides, lincosamides and fluoroquinolones. Resistance against 

other antimicrobials was not detected confirming that antimicrobial resistance does 

not yet pose a major problem for treatment of M. hyopneumoniae infections (Hannan 

et al., 1989; ter Laak et al., 1991; Inamoto et al., 1994; Hannan et al., 1997a,b). 

However, the rather high frequency of fluoroquinolone resistance is worrying and 

warrants prudent use of these antibiotics. 


This work was supported by the Federal Public Service of Public Health, Food 

Chain Security and Environment, Brussels, Belgium, grant no. S-6039. 

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11. Friis, N.F. (1975). Some recommendations concerning primary isolation of Mycoplasma 

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14. Gautier-Bouchardon, A.V., Reinhardt, A.K., Kobisch, M. & Kempf, I. (2002). In vitro 

development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in 

Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma synoviae. Veterinary 


15. Hannan, P.C.T. (2000). Guidelines and recommendations for antimicrobial minimum inhibitory 

concentration (MIC) testing against veterinary mycoplasma species. Veterinary Research 31, 

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17. Hannan, P.C.T., Windsor, H.M., de Jong A., Schmeer, N. & Stegemann, M. (1997a). 

Comparitive susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones. 

Antmicrobial Agents and Chemotherapy 41, 2037-2040. 

18. Hannan, P.C.T., Windsor, H.M. & Ripley, P.H. (1997b). In vitro susceptibilities of recent 

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(1996b). In vitro susceptibility of Mycoplasma hyosynoviae and M. hyorhinis to antimicrobial 

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23. Levisohn, S. (1981). Antibiotic sensitivity patterns in field isolates of Mycoplasma 

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quinolone resistance of Mycoplasma gallisepticum mutants obtained in vitro. Antimicrobial 


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30. Ter Laak, E.A., Noordergraaf, J.H. & Verschure, M.H. (1993). Susceptibilities of 

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31. Ter Laak, E.A., Pijpers, A., Noordergraaf, J.H., Schroevers, E.C. & Verheijden, J.H.M. 

(1991). Comparison of methods for in vitro testing of susceptibility of porcine mycoplasma 



of recent Belgian Mycoplasma bovis isolates. The Veterinary Record 153, 428-431. 

33. Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F. & de Kruif, A. (2002). 

Patterns of Mycoplasma hyopneumoniae infections in Belgian farrow-to-fiinish pig herds with 

diverging disease-course. Journal of Veterinary Medicine, Series B 49, 349-353. 

34. Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. & Haesebrouck, F. 

(2003). Evaluation of virulence of Mycoplasma hyopneumoniae field isolates. Veterinary 


35. Weisblum, B. (1995). Insights into erythromycin action from studies of its activity as inducer of 

resistance. Antmicrobial Agents and Chemotherapy 39, 797-805. 

36. Yamamoto, K. & Koshimizu, K. (1984). In vitro susceptibility of Mycoplasma hyopneumoniae 

to antibiotics, p. 116. Proceedings of the 8 th International Pig Veterinary Society Congress, 

Ghent, Belgium. 

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2.2.2 CHARACTERIZATION OF IN VIVO ACQUIRED 

RESISTANCE OF M. HYOPNEUMONIAE TO MACROLIDES 

AND LINCOSAMIDES 


CHARACTERISATION OF IN VIVO ACQUIRED RESISTANCE OF MYCOPLASMA HYOPNEUMONIAE TO 

MACROLIDES AND LINCOSAMIDES 

T. STAKENBORG, J. VICCA, P. BUTAYE, D. MAES, F.C. MINION, J. PEETERS, A. DE KRUIF & F. 

HAESEBROUCK 

MICROBIAL DRUG RESISTANCE 2005, 11, 291-295 

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2.2.2 RESISTANCE MECHANISM AGAINST MACROLIDES AND LINCOSAMIDES IN M. HYOPNEUMONIAE 

SUMMARY 

Macrolides and related antibiotics are used to control mycoplasma infections in 

the pig industry worldwide. Some porcine mycoplasmas, however, survive these 

treatments by acquiring resistance. The mechanism of acquired resistance to 

macrolides and lincosamides was studied in more detail for Mycoplasma 

hyopneumoniae (M. hyopneumoniae) by comparing both the phenotype and genotype 

of a resistant field isolate to 5 susceptible isolates. The minimal inhibitory 

concentrations (MICs) were significantly higher for the resistant isolate for all 

antibiotics tested. The MICs for the 16-membered macrolide tylosin ranged from 8 to 

16 µg for the resistant isolate and from 0.03 to 0.125 µg/ml for the 5 susceptible 

isolates. The MICs for the 15-membered macrolides and lincosamides were higher 

than 64 µg/ml for the resistant isolate while only 0.06 to 0.5 µg/ml for the susceptible 

isolates. M. hyopneumoniae isolates are intrinsically resistant to the 14-membered 

macrolides due to a G2057A transition (E. coli numbering) in their 23S rDNA. 

Therefore, high MICs were observed for all isolates, although the MICs for the 

resistant isolate were clearly increased. An additional, acquired A2058G point 

mutation was found in the 23S rRNA gene of the resistant isolate. No differences 

linked to resistance were found in the ribosomal proteins L4 and L22. The present 

study showed that 23S rRNA mutations resulting in resistance to macrolides and 

lincosamides as described in other Mycoplasma spp. also occur under field conditions 

in M. hyopneumoniae. 

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INTRODUCTION 

Mycoplasmas are the smallest free-living organisms, carrying no cell wall. As 

a consequence, they are naturally resistant to antibiotics interfering with cell wall 

synthesis. Additionally, a number of reports indicate a decrease in susceptibility of 

mycoplasmas against widely used antimicrobial agents, including the macrolides, 

lincosamides and streptogramins (MLS) (Hannan et al., 1997; Aarestrup and Friis, 

1998; Furneri et al., 2001; Okazaki et al., 2001; Thomas et al., 2003; Vicca et al., 

2004). MLS antibiotics have overlapping binding sites on the 23S rRNA and, hence, 

related antimicrobial activities. By binding to domain V of the 23S rRNA, they inhibit 

protein synthesis by means of blocking the path through which nascent peptides exit 

the ribosome (Retsema and Fu, 2001; Tenson et al., 2003). Additional studies have 

mapped the recognition site of the 14-membered macrolide erythromycin and its 

derived ketolides to domain II and IV of the 23S rRNA as well (Vester and 

Douthwaite, 2001; Berisio et al., 2003; Schlünzen et al., 2003). 

Bacterial species containing only 1 or 2 copies of rRNA genes, like all 

Mycoplasma species, tend to use mutations at bases 2057-2059 of the 23S rRNA as a 

way of acquiring resistance (Furneri et al., 2000; Hansen et al., 2002). Some 

mycoplasmas, like M. hyopneumoniae, are intrinsically resistant to 14-membered 

macrolides due to a G2057A transition in their 23S rDNA (Stemke et al., 1994; 

Ludwig et al., 1992; Furneri et al., 2000). Additional resistance to MLS antibiotics 

due to mutations at position 2609-2611 has been observed for several bacterial species 

(Garza-Ramos et al., 2001; Canu et al., 2002; Pereyre et al., 2002). A mutation at 

position 2062 was linked to resistance against josamycin, a 16-membered macrolide, 

in an in vitro selected M. hominis strain (Furneri et al., 2001; Hansen et al., 2002). For 

a number of other bacteria, mutations in the L4 and L22 binding proteins were linked 

to MLS resistance (Tait-Kamradt et al., 2000; Canu et al., 2002; Pihlajamaki et al., 

2002; Jones et al., 2003; Schlünzen et al., 2003; Farrell et al., 2004; Franceschi et al., 

2004; Pereyre et al., 2004). Nonetheless, acquired resistance to MLS in Mycoplasma 

species is rarely documented or is induced in vitro rather than observed in field 

isolates. The aim of this study was to fully characterize both phenotypically and 

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genetically the in vivo acquired resistance to macrolides and lincosamides in a M. 

hyopneumoniae field isolate. 


Media, bacterial isolates and antibiotics 

The F2, F5, F6, F18 and F19 isolates were all Belgian M. hyopneumoniae 

field-isolates obtained from lung tissue of fattening pigs collected at slaughterhouses. 

The J-strain (ATCC 25934) was used as a control strain. The Friis’ broth (Friis, 1975) 

without added antibiotics was used to grow the M. hyopneumoniae isolates. 

Erythromycin, tylosin and clindamycin were obtained from Sigma (UK). The 

other antibiotics were obtained from the drug's manufacturer as reference powder: 

lincomycin and azithromycin were kindly supplied by Pfizer (NY, USA), 

clarithromycin by Abott (Il, USA). 

The Minimal inhibitory concentration (MIC) test was performed using a 

macro broth dilution technique. Antibiotic stock solutions of 1,000 µg/ml were freshly 

prepared the day of use according to guidelines described by the National Committee 

for Clinical Laboratory Standards (NCCLS, 1999). Clarithromycin, erythromycin and 

azithromycin were dissolved in a minimum amount of ethanol and further diluted in 

water. All other antibiotics were directly dissolved in water. For each antimicrobial, 

two-fold dilutions ranging from 0.015 µg/ml to 64 µg/ml were prepared and each 

strain was tested twice. Broths were inoculated with the M. hyopneumoniae isolates, 

resulting in approximately 10 5 colour-changing units (CCU) per ml in a final volume 

of 2 ml. An additional tube contained only medium and was used as a negative 

control, while a second tube without antibiotics served as a positive control. The MIC 

was defined as the lowest concentration of antimicrobial agent in which no growth of 

M. hyopneumoniae was observed. Growth was detected by a colour change of the 

phenol red indicator from red to yellow due to glucose fermentation. The test was 

ended when no more colour change in the tubes was visible during two consecutive 

days. 

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DNA sequencing of the L4- and L22-genes and domain V of the 23S rDNA 

To sequence the L4 and L22 proteins, primers were designed based on the 

M. hyopneumoniae genome sequence of reference strain 232 (Minion et al., 2004). 

Primers for the amplification of domain V of the 23S rDNA were selected based on 

the M. hyopneumoniae ATCC 27719 strain (Genbank accession number X68421). All 

primers and reaction conditions used are listed in Table 1. After purification of the 

PCR product on Microcon 100 columns (Millipore, MA, USA), both strands were 

sequenced on a CEQ8000 sequencer (Beckmann, UK) according to the 

manufacturer’s instructions. The sequences were aligned for further analysis using 

Clustal W (V1.82). The sequences of domain V of the 23S rDNA of the M. 

hyopneumoniae isolates were compared with sequences of E. coli and other 

Mycoplasma species extracted from Genbank (Figure 1). 

Table 1: Selected primers used for the amplification and sequencing of the 23S, L4 

and L22 genes of the M. hyopneumoniae J-reference and field strains. 

Primer name Primer Sequence (5’ → 3’) Number of cycles (Cycle conditions) 1 

L4 FOR AGCATTCAAAGTCAGAAAAC 25 (30” 94°C; 30” 47.6°C; and 1’ 72°C) 

L4 REV 

GATTCTCTTCTCCAAATTAG 

L22 FOR AGCAGTCGCTTCACTCAAAA 25 (30” 94°C; 30” 52.5°C; and 1’ 72°C) 

L22 REV 

ACCTCTTTTTCTTGCGCTAA 

23S FOR GGTAGCGAAATTCCTTGTCA 25 (30” 94°C; 30” 55.2°C; and 1’ 72°C) 

23S REV 

GAGCAGCTCTCATCAATATTCC 

1 : All PCRs, unless stated otherwise, were performed using 2U of recombinant Taq polymerase, 5 µl of 

10X PCR buffer, 75 nmol MgCl2, 10 nmol of each dNTP and 10 pmol of both forward and reverse 

primer. 

- 130 -


|2040 |2050 |2060 |2070 |2080 |2090 |2100 

E. coli a GTACCCGCGG CAAGACGGAA AGACCCCGTG AACCTTTACT ATAGCTTGAC ACTGAACATT GAGCCTTGATG 

M. hyopneumoniae b TTACCCGCAT CAAGACGAAA AGACCCCGTG GAGCTTTACT ATAACTTCGT ATTGAGAATT GGTTTATTATG 

M. hyopneumoniae F19 TTACCCGCAT CAAGACGAGA AGACCCCGTG GAGCTTTACT ATAACTTCGT ATTGAGAATT GGTTTATTATG 

M. hominis AGACCCGCAT CTAGACGAAA AGACCCCGTG GAGCTTTACT ATAACTTCAT ATTGGAGTTT GATTTAACATG 

M. fermentans GTACCCGCAT CAAGACGAAA AGACCCCATG GAGCTTTACT ACAGTTTCGT ATTGGAACTT GGTCTAACATG 

M. genitalium TTAGGCGCAA CGGGACGGAA AGACCCCGTG AAGCTTTACT GTAGCTTAAT ATTGATCAAA ACACCACCATG 

M. pneumoniae TTAGGCGCAA CGGGACGGAA AGACCCCGTG AAGCTTTACT GTAGCTTAAT ATTGATCAGG ACATTATCATG 

M. gallisepticum TTAGGCGCAA CGGGACGGAA AGACCCCATG AAGCTTTACT GTAACTTAAT ATTGGGCAGA GTTTAGACATA 

M. penetrans TTAGGTGCGG TTAGACAAAA AGACCCCATG AAGCTTTACT GTAGCTTAAT ATTGGAAAAA TTTATTTCATT 

M. flocculare TTACCCGCAT CAAGACGAAA AGACCCCGTG GACGTTTACT ATAACTTCGT ATTGAGAATT GGTTTATTATG 

M. hyorhinis CGAACCGTAG TACGCTAAAA AGTGCCCGGA TGACTTGTGG ATAGC----- GGTGAAATTC CAATCGA-ACC 

M. pulmonis c CGAACCGTAG TACGCTGAAA AGTGCCCGGA TGACTTGTGA ATAGC----- GGTGAAATTC CAATCGA-ACC 

* * * * ** *** ** * ** * 

a 

Sequences were extracted from Genbank. Accession numbers are: Escherichia coli, ECO278710; M. 

hominis, AF443617; M. fermentans, AF422142; M. genitalium, U39634; M. pneumoniae, AE000007; 

M. gallisepticum, AE016968; M. penetrans, AP004174; M. flocculare, MYC16SR; M. hyorhinis, 

AF121891; M. pulmonis, AL445565. 

b 

No differences in the 23S DNA of isolate F2, F5, F6, F18, F23 and J were observed. 

c 

Resistance due to A2062 point mutations have been described elsewhere 10 

Figure 1: Multiple sequence-alignment of the 23S rDNA of different Mycoplasma 

spp. compared to E. coli. Nucleotides related to resistance to macrolides are presented 

in bold. The G2058A transition of the resistant M. hyopneumoniae F19 isolate is 

underlined as well. 

RESULTS 

Because the isolates grew at different rates, not all MICs were read on the 

same day. The J-reference strain grew faster than the other isolates, and the MICs of 

the antibiotics were reached after approximately 5 days, while for the other isolates it 

took up to one week. 

The MICs for the isolates are presented in Table 2. For each isolate, the MIC 

values of the replicates were equal to or differed from each other by only one dilution. 

The MICs for erythromycin and clarithromycin ranged from 8 – 32 µg/ml for all 

isolates tested, except for the resistant F19 isolate. For this isolate, the MIC values 

exceeded the highest concentration (64 µg/ml) tested. All isolates, except the F19 

isolate, were also susceptible to the other antibiotics tested. The MICs ranged from 

0.06 – 0.25 µg/ml for the 15-membered macrolide, azithromycin; from 0.03 – 0.125 

µg/ml for the 16-membered macrolide, tylosin; and from 0.125 – 0.5 µg/ml for the 

lincosamides, lincomycin and clindamycin. The MICs for the resistant isolate (F19) 

also exceeded the highest concentration of 64 µg/ml for these antibiotics, with the 

exception of tylosin (MICs ranging from 8-16 µg/ml). 

- 131 -


Sequence analysis of the 23S rDNA as shown in Figure 1, demonstrates the 

intrinsic resistance of certain Mycoplasma spp. to 14-membered macrolides due to a 

G2057A transition. An acquired A2058G transition was found exclusively in the 

resistant M. hyopneumoniae isolate. Only a small number of differences were present 

in the L22 proteins (>97% identity at DNA level). Protein L4 proved very conserved 

(>99% identical at DNA level) since no amino acid substitutions were found between 

any of the isolates (data not shown). 

Table 2: MICs (µg/ml) of MLS-antimicrobial agents for Belgian M. hyopneumoniae 

field isolates and the M. hyopneumoniae J reference strain obtained by the broth 

dilution test carried out in twofold. 

Isolate 

MIC (µg/ml) 

Azithromycin Tylosin Erythromycin Clarithromycin Clindamycin Lincomycin 

F2 0.125 a 0.03125 8 16 0.0625 0.25 

F5 0.125-0.25 0.03125 8 16 0.125-0.25 0.25 

F6 0.0625-0.125 0.03125 16 32 0.125 0.125 

F18 0.0625 0.0625 32 32 0.125-0.25 0.125 

F19 >64 8-16 >64 >64 >64 >64 

J 0.25 0.125 32 32 0.5 0.5 

a one value means that no difference between the repeated tests was observed. 

- 132 -


DISCUSSION 

Since M. hyopneumoniae isolates are difficult to grow on agar plates and 

colonies are hard to detect, a serial broth dilution technique using Friis’ medium was 

chosen to test for antibiotic resistance. This technique, based on an earlier report of 

Ter Laak et al. (1991), was performed in duplicate and proved to be reproducible. 

The J-reference strain seemed better adapted to the Friis’ medium and grew 

faster than the other isolates. This may explain the higher MICs, up to one dilution, 

for this strain. Nevertheless, all individual MICs were very similar for the susceptible 

isolates and clearly differed from those of the resistant F19 isolate. Moreover, MICs 

for the sensitive isolates were consistent with previous reports (Ter Laak et al., 1991; 

Tanner et al., 1993; Inamoto et al., 1994). 

Although M. hyopneumoniae isolates are naturally resistant to 14-membered 

macrolides due to a G2057A transition of the 23S gene (Vester and Douthwaite, 

2001), even higher MICs were observed for the resistant isolate. This increased 

resistance may well be explained by the observed A2058G transition since 

footprinting patterns examining the drug binding sites in other bacteria identified 

these specific nucleotides (Hansen et al., 1999; Edelstein, 2004). Hence, a lower 

affinity of the drugs to the 23S rRNA, due to the acquired mutation, results in a 

decreased antimicrobial activity. Apart from this increased insensitivity to 

erythromycin and clarithromycin, the F19 field isolate proved also resistant to all 

other antibiotics tested. This is in agreement with earlier reports in other bacterial 

species where mutations at position 2058 lead to high MLS resistant isolates (Vester 

and Douthwaite, 2001; Edelstein, 2004). This A2058G transition may even be the 

most frequently observed substitution in vivo in association with MLS resistance 

(Vester and Douthwaite, 2001) and was also found for clinical isolates of 

M. pneumoniae (Okazaki et al., 2001), although only limited data exist for in vivo 

acquired MLS resistance in Mycoplasma species. 

Recently, resistance to macrolides and lincosamides has been linked to 

modifications in the L4 and L22 rRNA-binding proteins (Gabashvili et al., 2001; 

O’Connor et al., 2004), especially in pneumococcal strains (Tait-Kamradt et al., 2000; 

Gabashvili et al., 2001; Canu et al., 2002; Pihlajamaki et al., 2002; Jones et al., 2003; 

- 133 -


Franceschi et al., 2004; O’Connor et al., 2004). In the present studies, none of the 

variations in these proteins were uniquely present in the resistant M. hyopneumoniae 

field isolate F19, and it is therefore unlikely that they are associated with acquired 

resistance. This is in agreement with an earlier report of resistant clinical strains of 

M. hominis and M. fermentans (Pereyre et al., 2002). 

In in vitro experiments, resistance to tylosin was obtained in M. hyopneumoniae 

isolates after only 7 or fewer passages in selective media (Hannan et al., 1997), 

indicating that acquired resistance due to mutations of the 23S RNA may occur quite 

fast for bacteria like M. hyopneumoniae, which only possess one copy of the rRNA 

operon (Stemke et al., 1994). The relation between the use of macrolides in pig 

rearing and the occurrence of acquired resistance has been indirectly demonstrated for 

M. hyosynoviae and other porcine bacteria (Aarestrup and Carstensen, 1998; 

Aarestrup and Friis, 1998; Butaye et al., 2001). In contrast, the prevalence of acquired 

resistance to macrolides for M. hyopneumoniae in the field is most likely low 

(Inamoto et al., 1994; Vicca et al., 2004). It is possible that resistant isolates do not 

spread easily and that occurrence of resistance remains localized in an area or even 

within a herd. Indeed, earlier RAPD data (Artiushin and Minion, 1996) and our own 

PFGE results (Stakenborg et al., 2005) show an enormous diversity between isolates 

from different farms, suggesting that clones do not readily spread in the environment, 

although further research on this issue is needed. Since the type of resistance 

described above is not encoded on a mobile element, and therefore not transferable 

between different isolates, the genetic stability of the mutation may also be an 

important factor. Although the A2058G transition has distinct advantages over the 

wild-type in the presence of macrolides, the situation may be different in the absence 

of the drugs (Vester and Douthwaite, 2001). In any event, the emergence of this 

resistance asks for a continuous monitoring, as it may have important therapeutic 

implications in the treatment of mycoplasma infections. 


This work was supported by a grant of the Federal Agency of Health, Food 

Chain Security and Environment (Grant number S-6136). 

The authors thank Sara Tistaert for skilful technical assistance. 

- 134 -


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2.2.3 MECHANISM OF RESISTANCE AGAINST THE 

FLUOROQUINOLONES FLUMEQUINE AND ENROFLOXACIN IN 

MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES 


MECHANISMS OF RESISTANCE AGAINST THE FLUOROQUINOLONES FLUMEQUINE AND 

ENROFLOXACIN IN MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES 

J. VICCA, D. MAES, T. STAKENBORG, P. BUTAYE, F.C. MINION, J. PEETERS, A. DE KRUIF & F. 

HAESEBROUCK 

IN PREPARATION 

139

2.2.3 MECHANISM OF RESISTANCE AGAINST FLUOROQUINOLONES OF M. HYOPNEUMONIAE ISOLATES 

SUMMARY 

Ten Mycoplasma hyopneumoniae field isolates that were either sensitive (5) or 

resistant (5) to the fluoroquinolones flumequine and enrofloxacin, two frequently used 

antibiotics in swine, were selected. Their quinolone resistance-determining regions 

(QRDR) of gyrA, gyrB, parC and parE were characterized. Parts of the DNA gyrase 

subunits, gyrA and gyrB, and the topoisomerase subunits, parC and parE, containing the 

QRDR, were sequenced. In all 5 resistant isolates one point mutation (C → A) in parC 

was found, resulting in an amino acid change from serine to tyrosine at position 80 (E. 

coli numbering). For 4 of these isolates, this was the only mutation found. These isolates 

had a MIC of enrofloxacin of 0.5 µg/ml, while for sensitive isolates the MIC of 

enrofloxacin was ≤ 0.06 µg/ml. One resistant isolate (Mh 20) had an extra mutation (C → 

T) in gyrA resulting in an amino acid change from alanine to valine at position 83 (E. coli 

numbering), correlated with an increase in the MIC of enrofloxacin (> 1 µg/ml). No 

mutations resulting in an amino acid change were detected in the QRDR of the gyrB and 

parE genes of the selected isolates. This is the first description of the mechanism of 

resistance against fluoroquinolones in M. hyopneumoniae. 

141


INTRODUCTION 

Mycoplasma hyopneumoniae is a major swine pathogen causing enzootic 

pneumonia, a chronic respiratory disease in pigs resulting in considerable economic 

losses. In a previous study (Vicca et al., 2004) conducted to determine the in vitro 

susceptibility of M. hyopneumoniae field isolates to frequently used antimicrobials in 

swine, 5 out of 21 isolates were found to be less susceptible or resistant to flumequine 

and enrofloxacin. This rather high frequency was unexpected since fluoroquinolone 

resistance does not often occur in swine respiratory pathogens (Hannan et al., 1997; 

Martel et al., 2001; Wallmann et al., 2003). 

Fluoroquinolones are broad-spectrum antimicrobials. Their use depends on the 

country regulations; fluoroquinolones are not allowed for use in pigs in the US but are 

allowed in the EU (World Health Organization, 1998). In Belgian pig herds, 

fluoroquinolones are frequently used as a prophylactic antibiotic during the suckling 

period mainly to prevent neonatal diarrhea (Timmerman et al., 2005). In older pigs, these 

antimicrobials are mainly used to parenterally treat individual animals with diarrhea, 

arthritis, meningitis or respiratory symptoms. The most frequently used fluoroquinolones 

in large animal veterinary medicine are flumequine and enrofloxacin. 

Fluoroquinolones are known to have two enzyme targets in the bacterial cell 

belonging to the topoisomerases type 2, namely DNA gyrase and topoisomerase IV. The 

first enzyme catalyses ATP-dependent negative supercoiling of DNA, the latter enzyme 

is essential for chromosome segregation (Gellert, 1981; Hooper, 2000). DNA gyrase is a 

tetramer composed of 2 GyrA and 2 GyrB subunits. Topoisomerase IV is similarly 

structured and is composed of 2 ParC and 2 ParE subunits. ParC is homologous to GyrA 

and ParE is homologous to GyrB. The primary target for fluoroquinolones in Gramnegative 

bacteria is the DNA gyrase, whereas in Gram-positive, including mycoplasmas, 

it seems to be the topoisomerase IV (Gellert, 1981; Belland et al., 1994; Khordusky et al., 

1995; Georgiou et al., 1996; Deguchi et al., 1997; Bébéar et al., 1998; Hooper, 2000; 

Beaucheron et al., 2004). However, some exceptions to this rule were found in 

Streptococcus pneumoniae and Mycoplasma hominis isolates for newer fluoroquinolones, 

such as sparfloxacin and gatifloxacin (Fukuda and Hiramatsu, 1999; Bébéar et al., 2000). 

For Mycoplasma gallisepticum, the preferential target of enrofloxacin is DNA gyrase 

(Reinhardt et al., 2002b). In several bacteria, mutations responsible for an increase in 

142


MIC-value were found in the subunits of these target genes (Yoshida et al., 1990; 

Yoshida et al., 1993; Bébéar et al., 1998; Bébéar et al., 1999; Reinhardt et al., 2002a). 

Resistance to fluoroquinolones has recently been described in M. hyopneumoniae 

(Vicca et al., 2004). The resistance mechanism, however, was unknown. In this study, the 

quinolone resistance determining regions (QRDR) of gyrA, gyrB, parC and parE were 

examined for the existence of point mutations related to resistance to fluoroquinolones. 



The ten M. hyopneumoniae field isolates selected for this study were obtained 

between 2000 and 2002 from slaughter pigs from 10 different Belgian farrow-to-finish 

pig herds and were previously used for MIC determination (Vicca et al., 2004). Isolate 

selection for this study was based on the MIC value: 5 isolates with the highest and 5 

isolates with the lowest MIC values for flumequine and enrofloxacin were retained. The 

MIC values of flumequine and enrofloxacin for 4 isolates (Mh 4, 8, 14 and 17) were > 16 

µg/ml and 0.5 µg/ml, respectively. For isolate Mh 20, the MIC of flumequine was > 16 

µg/ml and that of enrofloxacin > 1 µg/ml. The other five isolates (Mh 7, 10, 11, 15 and 

19) were susceptible to flumequine (MIC ≤ 2 µg/ml) and enrofloxacin (MIC ≤ 0.06 

µg/ml). 

DNA extraction and PCR amplification 

The M. hyopneumoniae isolates were grown in non-selective Friis medium and 

subsequently centrifuged at 5000 g for 10 minutes. DNA was extracted using the DNeasy 

Tissue kit (Qiagen, Westburg, Leusden, The Netherlands), according to the 

manufacturers’ instructions. Without further purification, an aliquot of the supernatant 

containing DNA was used as a template for PCR amplification. 

To sequence parts of the DNA gyrase subunits, gyrA and gyrB, and the 

topoisomerase subunits, parC and parE, containing the QRDR, primers were designed 

based on the M. hyopneumoniae genome sequence of reference strain 232 (Minion et al., 

2004). Oligonucleotides MhgyrAfor (5’-CTKCCRGATGTCCGWGATGG-3’)and 

MhgyrArev (5’-GTSGGRAARTCYGGCYCCGG-3’) were used to amplify a 557-bp 

143


gyrA fragment between positions 487 to 1043 (E. coli coordinates). A 937-bp gyrB 

fragment between positions 1994 to 3437 (E. coli coordinates) was amplified with 

primers MhgyrBfor (5’-ACATTCATAACCCTGAAGGC-3’) and MhgyrBrev (5’- 

GTCTCTCAAAGTTGTTCCGG-3’). To amplify the QRDR of parC, primers 

MhparCfor (5’-ATTCAGTAATTAATTCCCGG-3’) and MhparCrev (5’- 

TCTTCAAGGTAAATTTGCTG-3’) were selected to amplify a 1309-bp fragment 

between positions 19 to 1313 (E. coli coordinates) and a 735-bp parE fragment between 

positions 1046 to 1765 (E. coli coordinates) was amplified using the primers MhparEfor 

(5’-ATTCTTGAATTTGTTGGGC-3’) and MhparErev (5’- 

CCCAAGTCCTTTATAGCGC-3’). DNA amplification was performed with a DNA 

thermal cycler (model 9600 GeneAmp PCR system, Perkin-Elmer, Zaventem, Belgium). 

Each 50 µl PCR mixture contained 25 µl Mastermix (Invitrogen, Belgium), 2 µM of both 

primers and 2.5 µl DNA sample. Water was added to a total volume of 50 µl. For all 

amplification reactions, the same PCR running conditions were used, consisting of an 

initial cycle of 5 min denaturation at 94°C, followed by 35 cycles of 1 min denaturation 

at 94°C, 1 min of annealing at 55°C and 1 min of elongation at 72°C. After amplification, 

5 µl amplicon was mixed with 3 µl sample buffer (50% glycerol, 1mM cresol red). This 

mixture was electrophoresed in a 1.5% agarose gel for 75 min at 175 V in 0.5 x TBE 

(0.45 M Tris-HCl, 0.45 M boric acid, 0.01 M EDTA). 

Sequencing 

After purification of the PCR product with the Qiaquick PCR purification kit 

(Qiagen, Westburg, Leusden, The Netherlands), both strands of the PCR product were 

sequenced using the Big Dye Terminator v3-1 cycle sequencing kit (Applied Biosystems, 

Lennik, Belgium) on a ABI Prism 310 Genetic Analyser. The electropherograms were 

exported and converted to the sequence analysis software, Kodon ® (Applied Maths, Sint- 

Martens-Latem, Belgium). The nucleic acid sequences of the QRDR of gyrA, gyrB, parC 

and parE of the susceptible M. hyopneumoniae isolates were compared with those of the 

isolates with a MIC of > 16 µg/ml and ≥ 0.5 µg/ml for flumequine and enrofloxacin, 

respectively (Figure 1 and 2). The deduced amino acid sequences of susceptible isolate 

Mh 7 and resistant isolate Mh 20 isolate were compared with the sequences of 

Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli and to date fully 

sequenced human and veterinary Mycoplasma species (Figure 3). The percentage of 

144


identity between the susceptible Mh 7 isolate, the other organisms and the GenBank 

accession numbers are listed in Table 2. 

RESULTS 

PCR amplification and sequences of PCR products 

Each of the selected forward and reverse primer pairs amplified one PCR product. The 

deduced amino acid sequences of the partial gyrA, gyrB, parC, and parE region are 

presented in Figures 1 and 2. An acquired C264A transition (E. coli numbering) was 

found in the parC gene of all 5 isolates with MICs of > 16 µg/ml and ≥ 0.5 µg/ml for 

flumequine and enrofloxacin, respectively. This corresponds to an amino acid change 

from serine to tyrosine at position 80 (E. coli numbering). An additional transition was 

found in isolate Mh 20. The MIC of enrofloxacin for this isolate was > 1 µg/ml, while it 

was 0.5 µg/ml for the other resistant isolates. This additional transition, C635T, was 

found in gyrA, resulting in an amino acid change from alanine to valine at position 83 (E. 

coli numbering) (Table 1). In the same isolate, another substitution was found in gyrA: 

T630A. However, this substitution did not result in an amino acid change. Other silent 

substitutions in the QRDR of gyrA were found in isolate Mh 7 (G651A) and in isolates 

Mh 15, 19 and 20 (G759A). In the QRDR of gyrB, silent substitutions were found in 

isolates Mh 4, 8, 11, 14, 17 and 20 (T2529A) and isolates Mh 10 and 19 (G2577A). No 

silent substitutions were found in the QRDR of parC. In the QRDR of parE two silent 

substitutions were found: C315T in isolates Mh 7 and 15, and G345A in isolates Mh 7, 

11 and 15. The identity for the QRDR of the 4 fluoroquinolone target genes at DNA level 

was very high for all M. hyopneumoniae isolates; 96.30%, 98.80%, 98.78% and 97.53% 

for the QRDR of gyrA, gyrB, parC and parE, respectively. 

145


Table 1: MICs for flumequine and enrofloxacin and the amino acid mutations in gyrA 

and parC of the fluoroquinolone resistant field isolates of M. hyopneumoniae. 

MIC (µg/ml) 

Amino acid change (codon) 

Isolate Flumequine Enrofloxacin gyrA parC 

83 * 80 

Mh 7 2 0.06 - - 

Mh 10 2 0.06 - - 

Mh 11 1 0.03 - - 

Mh 15 2 0.06 - - 

Mh 19 2 0.06 - - 

Mh 4 > 16 0.5 - S(TCT)→Y(TAT) 

Mh 8 > 16 0.5 - S(TCT)→Y(TAT) 

Mh 14 > 16 0.5 - S(TCT)→Y(TAT) 

Mh 17 > 16 0.5 - S(TCT)→Y(TAT) 

Mh 20 > 16 > 1 A(GCT)→V(GTT) S(TCT)→Y(TAT) 

* : amino acid position according to E. coli numbering 

146


QRDR of gyrA 

Mh 4 517 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT 

Mh 7 GTCCACCGTCGAATTCTATATACAATGGCTGAACTCGGAATTACTTCGGGAACAAGTTATAAAAAATCCGCTAGAATTGTTGGTGATGTT 









Mh 4 607 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT 

Mh 7 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCAATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT 

Mh 8 CTTGGAAAATACCATCCTCATGGCGATGCTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT 







Mh 20 CTTGGAAAATACCATCCTCATGGAGATGTTTCTGTCTATGAATCGATGGTGCGAATGGCTCAACCTTTTTCCTTACGTTATCCTTTAGTT 

* * * 

Mh 4 697 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG 759 

Mh 7 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAG 





Mh 15 GATGGTCATGGAAATTTTGGATCAATTGATGGTGATGAAGCAGCAGCAATGCGTTATACAGAA 




* 

QRDR of gyrB 

Mh 4 2431 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG 

Mh 7 GCCGAACTTTATATTGTTGAGGGGAATTCAGCCGGGGGCAGTGCAAAAATGGGGCGTGACCGACATTTTCAGGCTATTTTACCTTTGCGG 









Mh 4 2521 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA 

Mh 7 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA 

Mh 8 GGAAAAGTCATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA 

Mh 10 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAAATTCTATCGATGATCACCGCTTTTGGAACAGGA 



Mh 15 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAGATTCTATCGATGATCACCGCTTTTGGAACAGGA 


Mh 19 GGAAAAGTTATTAATTCCCAACGGTTTCAGCTTGAAAAAGTACTTAAAAATGAAGAAATTCTATCGATGATCACCGCTTTTGGAACAGGA 


* * 

Mh 4 2611 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT 2679 

Mh 7 GTTGGTCCT---GAATTTGATATTAGTAAAATCCGCTATCAAAAAATAATTATTATGACGGATGCTGAT 









Figure 1: DNA sequence of the quinolone resistance determining region (QRDR) of the 

gyrA and gyrB subunit of DNA gyrase of 10 Mycoplasma hyopneumoniae field isolates. 

Silent mutations are underlined, substitutional mutations are underlined and bold. Nucleic 

acid position according to E. coli numbering. 

147


QRDR of parC 

Mh 4 146 

Mh 7 

Mh 8 

Mh 10 

Mh 11 

Mh 14 

Mh 15 

Mh 17 

Mh 19 

Mh 20 

GTTCAGAGGCGAATTCTTTATTCAATGTGACAGTTGGGGCTAAAAAATAGTAAAAATTACAAAAAATCTGCTAGAGTTGTCGGTGATGTA 










Mh 4 236 ATCGGAAAATATCATCCTCATGGTGATTATTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG 

Mh 7 ATCGGAAAATATCATCCTCATGGTGATTCTTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG 

Mh 8 ATCGGAAAATATCATCCTCATGGTGATTATTCAATCTATGATGCTCTTGTCAGACTTGCCCAGGAATGAAAAATGAACTCCCCGCTTGTG 








* 

Mh 4 326 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG 391 

Mh 7 GAAATGCACGGAAATAAAGGGTCAATTGATGA--CGATCCACCT-GCCGCGATGCGATATACAGAG 








QRDR of parE 

Mh 4 1298 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA 

Mh 7 CGCGAGCTTTTTTTGGTTGAAGGTGAATCGGCAGGTGGTTCTGCAAAACTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA 

Mh 8 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAGCTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA 


Mh 11 CGCGAGCTTTTTTTGGTCGAAGGTGAATCGGCAGGTGGTTCTGCAAAACTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA 


Mh 15 CGCGAGCTTTTTTTGGTTGAAGGTGAATCGGCAGGTGGTTCTGCAAAACTTGCAAGAAACCGTGAGTTTCAAGCAATTTTACCTTTAAAA 




* * 

Mh 4 1388 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA 

Mh 7 GGTAAAATTGTAAACGCCCAAAAAACAAGATTAATTGATCTATTAAAAAATGAGGAAATTATCGCAATTATTAGTGCATTAGGAACTGGA 









Mh 4 1478 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC 1540 

Mh 7 ATTGGTCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATCATGACTGACGCTGAC 



Mh 11 ATTGGCCAGAATTTTAACCTTAAGAACCTAAATTATGGCAAAATTATCATTATGACTGACGCTGAC 






* 

Figure 2: DNA sequence of the quinolone resistance determining region (QRDR) of the 

parC and parE subunit of topoisomerase IV of 10 Mycoplasma hyopneumoniae field 

isolates. Silent mutations are underlined, substitutional mutations are underlined and 

bold. Nucleic acid position according to E. coli numbering. 

148


DISCUSSION 

Resistance in M. hyopneumoniae field isolates was first described in a previous 

study where 5 of 21 isolates were found to be resistant to flumequine and less susceptible 

or resistant to enrofloxacin (Vicca et al., 2004). This rather high prevalence of 

fluoroquinolone resistance does not agree with the resistance rate found in other bacterial 

swine pathogens like Streptococcus suis (Martel et al., 2001; Aarestrup et al., 1998), 

Arcanobacterium pyogenes (Yoshimura et al., 2000), Pasteurella multocida and 

Mannheimia haemolytica (Wallmann et al., 2003). Mycoplasma hyosynoviae and 

Mycoplasma hyorhinis however, two other pathogenic mycoplasmas in swine, appear to 

exhibit a high resistance rate against flumequine (100% and 85% resistance, respectively) 

but are fully susceptible to enrofloxacin (Hannan et al., 1997). 

Several mechanisms for fluoroquinolone resistance have been described in 

different bacterial species. These include alterations in the two drug target enzymes, 

namely DNA gyrase and topoisomerase IV, changes in drug permeation through 

modifications in the outer membrane proteins, induction of active efflux systems, 

modifications in the peptidoglycan layer or the outer membrane proteins (Nikaido and 

Thanassi, 1993; Kaatz et al., 2002) and plasmid-correlated quinolone resistance 

(Martinéz-Martinéz et al., 1998; Hawkey, 2003). Although the existence of energy 

dependent efflux systems has recently been described for M. hominis (Raherison et al., 

2002), acquired resistance in mycoplasma species is usually due to alterations in the 

target enzymes. In the present study, the QRDR of the four target genes gyrA, gyrB, parC 

and parE were sequenced in fluoroquinolone susceptible and resistant M. hyopneumoniae 

isolates. The amino acid change at position 80 (E. coli numbering) in ParC, observed in 

all 5 resistant isolates, is the most common mutation related to fluoroquinolone resistance 

in Gram-positive bacteria (Ferrero et al., 1995; Ng et al., 1996; Yamagishi et al., 1996; 

Kanematsu et al., 1998), including M. hominis (Bébéar et al., 2003). For four of the M. 

hyopneumoniae isolates, this was the only mutation found and it resulted in at least an 8- 

fold increase in MIC of flumequine and enrofloxacin. Such isolates are considered to be 

resistant to flumequine (MIC > 16 µg/ml) (Hannan et al., 1997), while they are still 

considered to be susceptible to enrofloxacin (MIC = 0.5 µg/ml) (NCCLS, 2002). 

One isolate Mh 20 had an extra mutation (C → T) in gyrA at position 635, 

resulting in an amino acid change from alanine to valine at position 83 (E. coli 

149


numbering). This is associated with at least a 4-fold increase in MIC of enrofloxacin 

(MIC > 1 µg/ml) compared to isolates with only a mutation in parC. This location is 

another hot spot for fluoroquinolone resistance (Hooper, 1999). 

The occurrence of low-level resistance against fluoroquinolones after a single 

mutation in parC has earlier been described for Enterococcus faecalis, Staphylococcus 

aureus and Streptococcus pneumoniae, whereas high-level resistant isolates had 

mutations in both parC and gyrA (Kanematsu et al., 1998; Schmitz et al., 1998; Davies 

and Goldschmidt, 2002). In M. bovirhinis, however, a single mutation in parC (position 

80) resulted in different MIC profiles including low- and high-level resistant isolates 

(Hirose et al., 2004). The authors suggested that the differences in MIC might have been 

caused by the level of expression of the quinolone efflux transporter. 

As in fluoroquinolone resistant M. hominis, Ureaplasma urealyticum and 

Acholeplasma laidlawii isolates, no mutations were found in the QRDR of gyrB in M. 

hyopneumoniae. Such mutations have been described in in vitro selected resistant M. 

gallisepticum isolates (Reinhardt et al., 2002a). Also, no mutations resulting in amino 

acid changes were found in the QRDR of parE of the M. hyopneumoniae isolates. In 

clinical isolates of M. hominis however, a mutation resulting in an amino acid 

substitution in parE was previously observed (Bébéar et al., 1999). The absence of amino 

acid changes in GyrB and ParE of fluoroquinolone resistant M. hyopneumoniae isolates is 

in agreement with other studies reporting that amino acid changes in GyrB or ParE occur 

less frequently than in GyrA and ParC (Hooper, 1999). 

CONCLUSION 

Topoisomerase IV of M. hyopneumoniae seems the primary target for fluoroquinolones 

(flumequine and enrofloxacin) with position 80 in parC as the hot spot. A single mutation 

in parC is sufficient to reach resistance to flumequine, while a second mutation in the 

secondary target, DNA gyrase (gyrA), is necessary to make M. hyopneumoniae resistant 

to enrofloxacin. 


This work was supported by the Federal Public Service of Public Health, Food 

Chain Security and Environment, Brussels, Belgium, grant no. S-6039. 

150


151

GyrA 

Mh 7 

VHRRILYTMAELGITSGTSYKKSARIVGDVLGKYHPHGDASVYESMVRMAQPFSLRYPLVDGHGNFGSIDGDEAAAMRYTE 

Mh 20 VHRRILYTMAELGITSGTSYKKSARIVGDVLGKYHPHGDVSVYESMVRMAQPFSLRYPLVDGHGNFGSIDGDEAAAMRYTE 

Mhom 

VHRRILYGMSELGMFYTAPHKKSARIVGDVLGKYHPHGDSSVYEAMVRMAQDFSLRYPLIDGHGNFGSVDGDEAAAMRYTE 

Mgen 

VHRRVLYGAYIGGMHHDRPFKKSARIVGDVMSKFHPHGDMAIYDTMSRMAQDFSLRYLLIDGHGNFGSIDGDRPAAQRYTE 

Mgal 

PVTWVLYGAYTSGLTHDKPYRKSAQIVGHVMGKYHPHGDSAIYETMVRMAQPFSLRYMLIDGHGNFGSIDGDSAGAMRYTE 

Mmob 

VHRRILYGMTELGLFYNAQYKKSARVVGDVLGKYHPHGDSSVYEAMVRMAQDFSLRYPLIDGHGNFGSIDGDSAAAMRYTE 

Mmyc 

VHRRIIYAMNDLGITSDKPHKKSARIVGEVIGKYHPHGDSAVYETMVRMAQEFSYRYPLIDGHGNFGSIDGDGAAAMRYTE 

Mpen 

VHRRVLYAAYNMGMTHDKPYKKSARLVGEVIGKYHPHGDTAAYETMVRMAQDFSMRYMLVDGHGNFGSIDGDSAAAMRYTE 

Mpneu VHRRVLYGAYTGGMHHDRPFKKSARIVGDVMSKFHPHGDMAIYDTMSRMAQDFSLRYLLIDGHGNFGSIDGDRPAAQRYTE 

Mpul 

VHRRILYGMFDLGLHHSSAFKKSARIVGDVLGKYHPHGDASVYEAMVRMAQEFSMRYPLVDGHGNFGSIDGDPAAAMRYTE 

Saur 

VHRRILYGLNEQGMTPDKSYKKSARIVGDVMGKYHPHGDSSIYEAMVRMAQDFSYRYPLVDGQGNFGSMDGDGAAAMRYTE 

Spneu VHRRILYGMNELGVTPDKPHKKSARITGFVMGKYHPHGDSSIYEAMVRMAQWWSYRYMLVDGHGNFGSMDGDSAAAQRYTE 

Ecoli 44 VHRRVLYAMNVLGNDWNKAYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTE 

124 

** * *** * * * ***** * **** * ** * ** ***** *** * **** 

GyrB 

Mh 7 

AELYIVEGNSAGGSAKMGRDRHFQAILPLRGKVINSQRFQLEKVLKNEEILSMITAFGTGVGP-EFDISKIRYQKIIIMTDAD 

Mh 20 AELYIVEGNSAGGSAKMGRDRHFQAILPLRGKVINSQRFQLEKVLKNEEILSMITAFGTGVGP-EFDISKIRYQKIIIMTDAD 

Mhom 

RELFIVEGNSAGGSAKMGRDRSIQAILPLRGKVINAEKNSFASVLSNKEIATMIHALGTGINT-EFDINKLKYHKIIIMTDAD 

Mgen 

SELYIVEGDSAGGTAKTGRDRYFQAILPLRGKILNVEKSNFEQIFNNAEISALVMAIGCGIKP-DFELEKLRYSKIVIMTDAD 

Mgal 

SELYIVEGDSAGGSAKSGRDRFYQAILPLRGKVLNVEKANHEKIFKNEEIRTLITAIGAGVNP-EFSLDKIRYNKIIIMTDAD 

Mmob 

SELYIVEGNSAGGSAKMGRDRETQAILPLRGKVINAEKANIVKVFENTEILSLITALGTGVGP-EFNINKLRYHKVVIMTDAD 

Mmyc 

AELYLVEGDSAGGSAKTGRNRKFQAILPLRGKVLNVERVTEARAFSNNEIKSIITAIGTGIKE-ELDLSKLRYKKIVIMTDAD 

Mpen 

SELYIVEGDSAGGSAKLGRDRIFQAILPLKGKIINVEKAKSDKIFSNEEIINLITAIGAGVGP-EFKIDKLRYNKIILMTDAD 

Mpneu SELYIVEGDSAGGTAKTGRDRYFQAILPLRGKILNVEKSHFEQIFNNVEISALVMAVGCGIKP-DFELEKLRYNKIIIMTDAD 

Mpul 

SELYIVEGNSAGGSAKMGRDREFQAILPLRGKVINAEKNNIEKVFNNEEIQSLIIALGTGIAE-EFNINKLRYHKIIIMTDAD 

Saur 

CEIFLVEGDSAGGSTKSGRDSRTQAILPLRGKILNVEKARLDRILNNNEIRQMITAFGTGIGG-DFDLAKARYHKIVIMTDAD 

Spneu TELFIVEGDSAGGSAKSGRNREFQAILPIRGKILNVEKASMDKILANEEIRSLFTAMGTGFGA-EFDVSKARYQKLVLMTDAD 

Ecoli 418 SELYLVEGDSAGGSAKQGRNRKNQAILPLKGKILNVEKARFDKMLSSQEVATLITALGCGIGRDEYNPDKLRYHSIIIMTDAD 500 

* *** **** * ** ****** ** * * * * ** ***** 

ParC 

Mh 7 

VQRRILYSMWQLGLKNSKNYKKSARVVGDVIGKYHPHGDSSIYDALVRLAQEWKMNSPLVEMHGNKGSIDD-DPPAAMRYTE 

Mh 20 VQRRILYSMWQLGLKNSKNYKKSARVVGDVIGKYHPHGDYSIYDALVRLAQEWKMNSPLVEMHGNKGSIDD-DPPAAMRYTE 

Mhom 

VQRRILYSMWNLHLKNSEPFKKSARIVGDVIGRYHPHGDSSIYEALVRMAQDWKSNFPLIEMHGNKGSIDD-DPAAAMRYTE 

Mgen 

VQRRILYGMFQMGLKPTTPYKKSARAVGEIMGKYHPHGDSSIYDAIIRMSQSWKNNWTTVSIHGNNGSVDG-DNAAAMRYTE 

Mgal 

VQRRVLYGMYNLGLYYNKSYRKSAATVGEVIGKFHPHGDSSIYEALVRMTQSWKNNIPLIDMQGNNGSIDG-DNAAAMRYTE 

Mmob 

VQRRILYSMHELGLTSEKPFKKSARVVGDVIGKYHPHGDSSIYEAMVRMSQEWKMNVPLIQMHGNIGSIDD-DPAAAMRYTE 

Mmyc 

VQRRILYAMNQLNLTFDKPYKKSARVVGEVIGKYHPHGDSSIYDAMVRMSQWWKVNIPLVDMQGNNGSIDG-DSAAAMRYTE 

Mpen 

VQRRILHAMNELKIHHDKPYKKSARTVGEVIGKYHPHGDSSIYEAMVRMSQEWKNNLPLLDMQGNKGSIDG-DSPAAMRYTE 

Mpneu VQRRILYGMYQMGLKPTSPYKKSARAVGEIMGKYHPHGDASIYDAIVRMSQAWKNNLTTISIHGNNGSIDG-DNAAAMRYTE 

Mpul 

VQRRILYSMMDLKLWNDKPFKKSARIVGDVIGKYHPHGDSSIYEAMVRMAQDWKMNIPLIQMHGNIGSIDD-DPAAAMRYTE 

Saur 

VQRRILYAMYSSGNTHDKNFRKSAKTVGDVIGQYHPHGDSSVYEAMVRLSQDWKLRHVLIEMHGNNGSIDN-DPPAAMRYTE 

Spneu VQRRILYSMNKDSNTFDKSYRKSAKSVGNIMGNFHPHGDSSIYDAMVRMSQNWKNREILVEMHGNNGSMDG-DPPAAMRYTE 

Ecoli 41 VQRRIVYAMSELGLNASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLVDGQGNWGAPDDPKSFAAMRYTE 

122 

**** * *** ** * ***** * * * ** * ******* 

ParE 

Mh 7 

Mh 20 

Mhom 

Mgen 

Mgal 

Mmob 

Mmyc 

Mpen 

Mpneu 

Mpul 

Saur 

Spneu 

Ecoli 

RELFLVEGESAGGSAKLARNREFQAILPLKGKIVNAQKTR-LIDLLKNEEIIAIISALGTGIGQNFNLKNLNYGKIIIMTDAD 

RELFLVEGESAGGSAKLARNREFQAILPLKGKIVNAQKTR-LIDLLKNEEIIAIISALGTGIGQNFNLKNLNYGKIIIMTDAD 

KELFLVEGDSAGGSAKLGRNRVTQAILPLRGKVINTDKAK-LSDVLANEEIATIINTIGAGIGEDFNLKNAQYHKIIIMTDAD 

KELFIVEGDSAGGTAKMGRDRIFQAILPLRGKVLNVEKINNKKEAITNEEILTLIFCIGTGILTNFNIKDLKYGKIIIMTDAD 

NEIFLVEGDSAGGTAKSGRDKRFQAILPLRGKVVNVEKSR-LQDLLKNEEILSIISCLGCGIGNNFNIKNLKYHKIIIMTDAD 

KELFLVEGDSAGGSAKLGRNSQFQAILPLKGKVINCEKTK-LIDVLKNEEISTIINTIGAGIGADFDLKKANYHKVVIMTDAD 

NELYLVEGDSAGGSAKTGRNRKFQAILPLRGKVINSEKAK-LVDLLKNEEIQSIINAIGAGVGKDFDISDINYGKIIIMTDAD 

TELFLVEGDSAGGSAKLGRDKRYQAILPLKGKVINVEKAM-LKDLLKNEEISTIISSIGGSIGSDFCLPDVKYSKIIIMTDAD 

RELFVVEGDSAGGTAKMGRDRFLQAILPLRGKVLNVEKINNKKEAINNEELLTLIFCIGTGIGNNFTIRDRKYDKIIIMTDAD 

NEIFLVEGDSAGGSAKSGRNRRFQAILPLRGKVINTEKSR-LIDILKNEEISTIINALNTGVGEDFEIKNLKYHKIIIMTDAD 

NELYLVEGDSAGGSAKLGRDRKFQAILPLRGKVINTEKAR-LEDIFKNEEINTIIHTIGAGVGTDFKIEDSNYNRVIIMTDAD 

NELYLVEGDSAGGSAKQGRDRKFQAILPLRGKVINTAKAK-MADILKNEEINTMIYTIGAGVGADFSIEDANYDKIIIMTDAD 

412 TELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVS-SDEVLASQEVHDISVAIGIDPDSD-DLSQLRYGKICILADAD 

492 

* *** **** ** * *** ** ** * * * * *** 

Figure 3: Alignment of Mycoplasma hyopneumoniae GyrA, GyrB, ParC and ParE 

QRDR amino acid sequences of a susceptible isolate (Mh 7) with its counterpart from 

M. hyopneumoniae (resistant isolate, Mh 20), M. hominis (Mhom), M. genitalium 

(Mgen), M. gallisepticum (Mgal), M. mobile (Mmob), M. mycoides subsp. mycoides 

SC (Mmyc), M. penetrans (Mpen), M. pneumoniae (Mpneu), M. pulmonis (Mpul), 

Staphylococcus aureus (Saur), Streptococcus pneumoniae (Spneu) and Escherichia 

- 152 -


coli (Ecoli). GenBank accession numbers and percentage homology are listed in Table 

2. An asterisk indicates a residue identical in all 13 amino acid sequences. Dashes 

indicate gaps. 

- 153 -


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Haesebrouck, F. (2001). Prevalence and mechanism of resistance against macrolides and 

lincosamides in Streptococcus suis isolates. Veterinary Microbiology 83, 287-297. 

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B 47, 139-143. 

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2.2.4 THE EFFICACY OF TYLOSIN PREMIX FOR THE 

TREATMENT AND CONTROL OF MYCOPLASMA 

HYOPNEUMONIAE INFECTIONS 


EFFICACY OF IN-FEED MEDICATION WITH TYLOSIN FOR THE TREATMENT AND CONTROL OF 

MYCOPLASMA HYOPNEUMONIAE INFECTIONS 

J. VICCA, D. MAES, L. JONKER, A. DE KRUIF & F. HAESEBROUCK 

THE VETERINARY RECORD 2005, 156, 606-610 

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2.2.4 EFFICACY OF TYLOSIN FOR TREATMENT AND CONTROL OF M. HYOPNEUMONIAE INFECTIONS 

SUMMARY 

The efficacy of in-feed medication with tylosin (Tylan ® Premix; Elanco 

Animal Health) for the treatment of enzootic pneumonia was examined in an 

experimental Mycoplasma hyopneumoniae infection model. Three groups of 10 

conventional M. hyopneumoniae-free piglets were inoculated intratracheally with a 

highly virulent M. hyopneumoniae field isolate (non-treated control (non-TP) group 

and tylosin premix (TP) group) or with sterile culture medium (non-infected and nontreated 

control (NC) group). Twelve days after infection, tylosin treatment of the TP 

group was initiated (100 mg/kg of feed for 21 days). Animals were daily examined for 

the presence of clinical signs and a respiratory disease score (RDS) was given per pig. 

Thirty-three days after infection, all pigs were euthanased and the lung lesions were 

quantified. Lung samples were processed for immunofluorescence testing for M. 

hyopneumoniae. Average RDS and lung lesion scores were significantly higher 

(P


INTRODUCTION 

Enzootic pneumonia, with Mycoplasma hyopneumoniae as the primary agent, 

remains an important health disorder in modern pig herds. Enzootic pneumonia has 

been demonstrated to affect animal welfare and to cause substantial economic losses, 

either directly or by potentiating and increasing the susceptibility of the lungs to 

secondary pathogens like Pasteurella multocida and Actinobacillus 

pleuropneumoniae (Maes et al., 1999a, 2000). M. hyopneumoniae also plays a key 

role in porcine respiratory disease complex, together with porcine reproductive and 

respiratory syndrome virus, swine influenza virus and other pathogens. 

Control of enzootic pneumonia is largely based on good management 

practices, optimal housing conditions, vaccination and the use of antibiotics. 

Commercial M. hyopneumoniae vaccines are widely used and several studies have 

shown that vaccination has beneficial effects on daily weight gain (DWG) and 

mycoplasmal pneumonia lesions (Dohoo and Montgomery, 1996; Maes et al., 1998, 

1999b). Compared to vaccination, the use of antibiotics in M. hyopneumoniae 

outbreaks has the advantage that they can be applied in a more flexible way and that 

they can also decrease infections with other (respiratory) bacterial pathogens. 

Across the European Union, tylosin is used for the treatment and control of 

enzootic pneumonia in pigs through administration via the feed. Tylosin is a 

macrolide drug developed in 1961 and is exclusively used in veterinary medicine. 

Macrolides inhibit bacterial protein synthesis by reversibly binding to the 23 S 

ribosomal RNA in the 50 S subunit of the ribosome and bind at the donor site, thus 

preventing the translocation necessary to keep the peptide chain growing (Brisson- 

Noël et al., 1988). Tylosin, like other macrolides, is widely distributed throughout 

tissues and body fluids (Prats et al., 2002). 

Studies have demonstrated good efficacy of tylosin against M. hyopneumoniae 

in vitro (Ter Laak et al., 1991; Tanner et al., 1993; Wu et al., 1997) and in vivo 

(Hannan et al., 1982; Ueda et al., 1994). Previous in vivo studies were conducted with 

neonatal piglets inoculated with lung homogenate containing different Mycoplasma 

species or the preventive use of tylosin was examined. The current study was 

conducted in order to further examine the efficacy of tylosin when applied at the onset 

of clinical symptoms following infection with M. hyopneumoniae. Therefore, pigs 

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were experimentally infected with a high dosis (7 ml of 10 7 CCU/ml) of a highly 

virulent M. hyopneumoniae field isolate. The currently licensed dosage of 100 mg of 

tylosin per kg feed for 21 days was applied. 


M. hyopneumoniae isolate 

For the inoculation of the piglets, a Belgian M. hyopneumoniae field isolate 

was used. The isolate was obtained in 2000 from a pig herd experiencing clinical 

symptoms associated with enzootic pneumonia and could be filter cloned after 10 

passages in vitro. The isolate proved to be highly virulent during earlier comparative 

virulence studies (Vicca et al., 2003). The minimal inhibitory concentration of tylosin 

for this isolate was


piglets was housed in rooms equipped with absolute filters (HEPA U15) to avoid 

spread of M. hyopneumoniae-organisms between groups. 

Immediately after weighing and allocation to the treatment groups, all pigs 

were anaesthetized intramuscularly with 0.22 ml/kg of a mixture of xylazin (Xyl-M ® 

2%; VMD) and Zolazepam and Tiletamin (Zoletil ® 100; Virbac), ear tagged and 

inoculated intratracheally with 7 x 10 7 CCU of the M. hyopneumoniae isolate in 7 ml 

inoculum (non-treated control (non-TP) group and tylosin premix (TP) group) or with 

7 ml sterile culture medium (non-infected and non-treated control (NC) group). The 

inoculation day was designated as day 0 after infection. 

The study was conducted after approval by the Ethical Committee for animal 

experiments of the Faculty of Veterinary Medicine, Ghent University. 

Tylosin treatment 

None of the groups received any antimicrobials during the premedication 

period (0-12 days after infection). Tylosin treatment started at 12 days after infection, 

the day after onset of the disease (11 days after infection) when 10% of the animals 

over the two M. hyopneumoniae inoculated groups showed coughing. The duration of 

the medication period was 21 days (13-33 days after infection). 

Blank unmedicated feed was given to the non-TP and NC group. For the TP 

group, a commercial batch of Tylan ® 100 Premix (tylosin 10%, 100 g/kg premix; 

Elanco Animal Health) was used. One gram Tylan ® Premix was mixed per kg blank 

unmedicated feed, this was equivalent to approximately 3-6 mg tylosin/kg 

bodyweight. At the beginning of the study, the presence of the appropriate tylosin 

level in a pooled sample was confirmed to be 99.6 mg/kg feed. 

Clinical evaluation and performance parameters 

For a period of 33 days following challenge, pigs were observed daily for 25 

minutes, to evaluate demeanour, abdominal filling (gauntness), presence of dyspnoea 

and tachypnoea and behavioural changes. In addition, a clinical respiratory disease 

score (RDS) was assessed daily from 0 to 33 days after infection (Halbur et al., 1996). 

RDS scores could range from 0 to 6: 0 (no coughing), 1 (mild coughing after 

encouraged movement), 2 (mild coughing while leaving the pigs undisturbed), 3 

- 162 -


(moderate coughing after encouraged movement), 4 (moderate coughing while 

leaving the pigs undisturbed), 5 (severe coughing after encouraged movement), 6 

(severe coughing while leaving the pigs undisturbed). The number of coughing days 

was assessed as the total number of days on which at least one pig was coughing. 

Rectal temperatures were measured on a daily basis from 0-10 days after 

infection and every other day from 12-33 days after infection. 

To calculate average daily weight gain, the individual bodyweight of all 

animals was obtained on 0 days after infection, 7 days after infection, at the start of 

the medication period (12 days after infection), 19 days after infection, 22 days after 

infection and 33 days after infection. To be able to calculate the feed intake and feed 

conversion ratio per treatment group, feed allocations were recorded daily from 0 

days after infection onwards throughout the duration of the study. Feed leftovers were 

removed and weighed on the mentioned pig weighing days. 

Post-mortem examinations 

All pigs were euthanased at 33 days after infection by deep anesthaesia with 

0.3 ml/kg of a mixture of xylazine (Xylm ® 2%; Intervet) and zolazepam and tiletamin 

(Zoletil ® 100; Virbac) followed by exsanguination. The lungs were removed and 

macroscopic pneumonia lesions were quantified using a lung lesion score diagram 

(Hannan et al., 1982). Total lung lesion scores could vary between score 0 (no 

lesions) and a theoretical maximum of 35 (100% of the lung area affected by 

pneumonia). A digital picture (DSC-S50; Sony Cyber-shot) was taken from every 

lung for future reference. 

Two lung tissue samples were collected from every lung from sections with 

lesions typical for M. hyopneumoniae infection (preferably the cardiac lobes) or from 

healthy tissue when macroscopical lesions were not apparent. The 2 samples were 

processed in a semi-quantitative immunofluorescence assay (score 0-3) to detect the 

presence of M. hyopneumoniae (Kobisch et al., 1978); 0: (no immunofluorescence), 1, 

2 and 3 (limited, moderate and intense immunofluorescence, respectively). An 

additional lung tissue sample per pig was taken for bacteriological examination. 

Twenty percent (w/v) suspensions of lungs were made, inoculated on Columbia agar 

supplemented with 5 percent sheep blood (blood agar) and incubated at 37°C for 48 

hours to check the presence of secondary bacteria in the lungs. The plates were cross- 

- 163 -


streaked with Staphylococcus aureus for support of Actinobacillus pleuropneumoniae 

growth. 

To demonstrate the presence and localisation of tylosin in the lung, entire 

transverse sections from across the bottom of the diaphragmatic lobe of the right lung 

were collected. After sampling, the tissues were immersed in paraformaldehyde 

fixative for one month and sent to the Veterinary Laboratory Agency (VLA), New 

Haw, Addlestone, Surrey, UK for further processing. Each sample was cut into 4 

pieces and processed to paraffin wax embedded blocks. Immunostained sections were 

examined by light microscopy and subjectively assessed for specific cellular tylosin 

staining and diffuse non specific background staining. Digital photomicrographs 

(DX1200; Nikon Digital Camera, Nikon ACT-1 software) of representative specific 

immunodetection in each experimental group were produced. 

Serology for M. hyopneumoniae 

A blood sample of every pig was taken at 0 and 33 days after infection to 

detect serum antibodies against M. hyopneumoniae, using the DAKO ® Mh ELISA 

(DAKO, Glostrup, Denmark) (Feld et al., 1992). Sera with optical density (OD)- 

values


test was used to compare the number of animals between groups with a positive M. 

hyopneumoniae serology result and with a positive IF score. Statistical analyses were 

performed using SAS 8 (SAS Institute, 1999). 

RESULTS 

Clinical and performance parameters 

One animal in the NC group, two animals in the non-TP group and three 

animals in the TP group were found with arthritis, presumably caused by 

Streptococcus suis. They were intramuscularly injected 3 times with amoxycillin, 

every other day. One animal in the negative control group died during blood sampling 

on the day of infection. No other animals were removed from study. 

During the pre-medication period, coughing was rarely observed in the 3 

groups. For the entire study period, coughing was present in both groups inoculated 

with M. hyopneumoniae but was rarely observed in the NC group (Figure 1). 

Coughing started at approximately 11 days after infection and increased steadily 

towards the end of the study. Average RDS were significantly higher (P


2.5 

2 

Average RDS 

1.5 

1 

0.5 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 

DAI 

0 DAI 

infection 

13 DAI 

start tylosin treatment 

23 DAI 

33 DAI 

euthanasia 

pre-treatment period 

tylosin treatment period 

NC TP non-TP 

Figure 1: Average respiratory disease score (RDS) of the M. hyopneumoniae infected 

and tylosin treated group (TP) (n= 10 pigs), the M. hyopneumoniae infected and nontreated 

group (non-TP) (n= 10 pigs) and the non-infected and non-treated control 

(NC) group (n= 9 pigs), during the pre-treatment period (0-12 days after infection 

(DAI)) and the tylosin treatment period (13-33 DAI). 

The average rectal temperatures were not significantly different between the 

treatment groups (average temperatures were 39.5, 39.6 and 39.7 °C in the NC, non- 

TP and TP group, respectively). Rectal temperatures slowly decreased towards the 

end of the study in all groups. 

None of the performance parameters were significantly different between the 

study groups. The average weights (SD) for the NC, non-TP and TP group were 8.18 

(1.21), 8.07 (1.08) and 7.89 (1.42) kg at the beginning of the study, 8.85 (1.25), 9.70 

(1.38) and 9.42 (1.90) at 7 days after infection, 11.15 (1.55), 11.10 (1.54) and 11.10 

(2.30) at the start of the medication period (12 days after infection), 14.46 (1.95), 

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15.44 (2.39) and 14.34 (3.36) at 19 days after infection, 16.38 (2.53), 17.14 (2.72) and 

15.98 (3.74) at 22 days after infection and 23.87 (3.26), 23.19 (4.49) and 23.70 (5.05) 

kg at 33 days after infection, respectively. For the NC, non-TP and TP group, the 

ADG was 0.48 (0.08), 0.46 (0.11) and 0.48 (0.12) kg, the average daily feed intake 

per pig was 0.79, 0.80 and 0.78 kg and the FCR 1.64, 1.75 and 1.64, respectively. 

In the tylosin treatment group, the nominal intake of tylosin during the 

medication period was 5.7 mg/kg bodyweight, which was within the target intake 

range of 3-6 mg/kg bodyweight. 

Post-mortem examinations (Table 1) 

The proportion of animals with lung lesions was significantly higher (P


A 

B 

Figure 2: Digital photographs of immunostained, paraffin embedded lung tissue 

sections, magnification x400. A: section of a pig of the non-infected, non-treated 

control group. B: section of a pig of the infected, tylosin treated group. Infiltrating 

macrophages show cytoplasmic tylosin (arrows). 

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Serology for M. hyopneumoniae (Table 1) 

None of the 30 pigs had a positive ELISA result at 0 days after infection. The 

proportion of animals that seroconverted at 33 days after infection was significantly 

higher (P


DISCUSSION 

In the swine industry, antibiotics are frequently used to prevent and treat 

diseases such as enzootic pneumonia, one of the most important respiratory diseases 

in swine worldwide. Outbreaks of enzootic pneumonia still occur frequently in 

intensive pig production systems and the onset cannot always be predicted. For those 

outbreaks, antibiotics are necessary to limit the number of diseased animals and the 

extent of the lesions and consequently to improve better animal welfare and limit the 

economical loss. 

Tylosin is one of the oldest antibiotics for veterinary medicine that is still 

frequently used (Chauvin et al., 2002). A previous study demonstrating the efficacy of 

tylosin for the treatment of enzootic pneumonia was carried out with neonatal piglets 

inoculated intranasally with a lung homogenate containing different mycoplasma 

species. Treatment with tylosin tartrate was initiated at 14 days after infection and 

lasted for 10 days (Hannan et al., 1982). In other studies, the preventive use of tylosin 

against M. hyopneumoniae infections was examined (Ueda et al., 1994). In our study, 

the efficacy of tylosin was evaluated at the onset of an enzootic pneumonia outbreak. 

Tylosin treatment only started at 12 days after infection, when approximately 10% of 

the pigs were coughing. In this way, the study tried to simulate field conditions where 

a treatment is often initiated when 10-20% of the pigs are coughing. 

After oral treatment, tylosin has been shown to be widely distributed in pigs 

throughout body fluids and the drug also reaches peripheral tissues (Prats et al., 

2002). In the present study, strong immunostaining was detected in the cytoplasm of 

lung alveolar macrophages of pigs treated with tylosin, confirming that this antibiotic 

accumulates in these cells (Scorneaux and Shryock, 1998). 

Respiratory tract disease symptoms and lung lesions were less extensive and 

less severe in the tylosin treated group than in the non-treated control group, 

demonstrating the efficacy of this antibiotic to treat enzootic pneumonia in swine 

inoculated with a high dose of a highly virulent M. hyopneumoniae isolate. The 

results of the semi-quantitative immunofluorescence indicated that tylosin treatment 

may decrease the number of M. hyopneumoniae organisms in the lungs, although 

average IF scores were not significantly different between the TP group and non-TP 

group. Kobisch et al., (1978) and Amanfu et al., (1984) showed that the intensity of 

- 170 -


immunofluorescence has indeed been correlated with the number of organisms in the 

lungs. Secondary bacterial lung infections were only detected in 2 pigs of the nontreated 

positive control group. In severe field cases of enzootic pneumonia, the 

frequency of secondary bacterial infections is usually higher (Maes et al., 1998). The 

reason for this is not clear but may be due to different housing and management 

conditions (Clark et al., 1991). In our study, all pigs were simultaneously inoculated 

with a high dose of M. hyopneumoniae. In the field, the infection dose is not known 

but due to the chronic nature of the disease, repeated infections can be expected and 

the infection of the pigs might be spread over time. Stocking density, ventilation and 

hygienic measures may also differ substantially from the circumstances under 

experimental conditions. Nevertheless, experimental studies remain important to 

precisely evaluate the efficacy of antibiotics since in contrast with field studies, 

variables that are out of the control of the investigator are not likely to influence the 

results. 

CONCLUSION 

Tylosin administered via the feed at a level of 100 mg/kg feed for 21 days was 

efficacious for the treatment of established mycoplasmal disease. Even if M. 

hyopneumoniae vaccination is widely used in modern pig herds, it remains important 

to have effective, easily applicable antibiotics which can be used at the moment of 

acute outbreaks of enzootic pneumonia. 


This work was supported by a grant from Elanco Animal Health Benelux, a 

Divison of Eli Lilly and Co. 

The authors are grateful to Mrs. Y. Spencer from the VLA, New Haw, 

Addlestone, Surrey, UK, for providing the figures demonstrating the presence of 

tylosin in the alveolar macrophages. 

- 171 -


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3 GENERAL DISCUSSION

3 GENERAL DISCUSSION 

Infections with M. hyopneumoniae, the primary agent of enzootic pneumonia 

in pigs, are very common worldwide, especially in countries with intensive 

production systems. Enzootic pneumonia causes major economic losses to the pig 

industry due to decreased daily weight gain, increased feed conversion ratio, number 

of days to reach slaughter weight and medication costs. Losses in the Belgian pig 

industry are estimated to be at least 14 million euro each year. Studying the factors 

influencing the disease-course, which may evolve either subclinically or clinically, is 

essential to limit or reduce these losses. The best studied factor is the influence of the 

environment on the disease outcome. Poor management and housing conditions have 

been considered to be important deteriorating factors (Clark et al., 1991; Maes et al., 

1996; Ross, 1999). The host susceptibility, in contrast, is not found to be a major 

contributing factor (Goodwin et al., 1969; Piffer and Ross, 1984; Kobisch et al., 1993; 

Wallgren et al., 1998). The third factor that may influence the disease-course is 

related to the organism causing the disease. Antigenic and genetic diversity of M. 

hyopneumoniae isolates has been reported earlier (Ro and Ross, 1983; Frey et al., 

1992; Artiushin and Minion, 1996) but no correlation was made with the virulence. In 

this thesis, differences in virulence between M. hyopneumoniae field isolates were 

studied. 

In a first field study (chapter 2.1.1), herds with either poor housing and 

management conditions but experiencing subclinical disease or herds with good 

housing and management conditions but with a clinical disease-course were selected. 

This selection procedure was used to increase the chance of isolating low and high 

virulence M. hyopneumoniae isolates. 

Six isolates obtained in the first study were used in an experimental infection 

model (chapter 2.1.2) to asses the virulence. This model proved to be highly 

reproducible and can be used to evaluate the efficacy of vaccins or antimicrobial 

treatment. It also allows classifying the isolates as low, moderately and highly 

virulent. 

Animals inoculated with a high virulence isolate developed more severe lung 

lesions. In the lungs of these animals, the immunofluorescence score was significantly 

higher than in the lungs of animals inoculated with a low virulence strain. This 

indicates that a higher number of M. hyopneumoniae organisms in the lungs results in 

more severe lung lesions. The higher immunofluorescence score can be due to a faster 

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multiplication in the host, a better circumvention or modulation of the host immune 

response or a more efficient adherence of high virulence isolates. A difference in 

capability to attach to cilia between M. hyopneumoniae strains has been demonstrated 

by Minion et al. (2000) and was correlated with the number of repeat regions in RR1 

of the p97 protein of M. hyopneumoniae. In that study, a minimum of eight repeats 

was necessary to make the M. hyopneumoniae organisms able to attach to the cilia. 

Recently, a difference in the number of repeats in RR1 was detected in our isolates (T. 

Stakenborg, personal communication, 2005) but no correlation was found between the 

number of repeats and the virulence of the isolates. 

Using the OPA-3 primer (5’ AGTCAGCCAC 3’) (Artiushin and Minion, 

1996), a RAPD band of about 5 kbp was only found in high and moderate virulence 

isolates, but not in low virulence isolates. In further studies, this RAPD band was 

sequenced (accession n° AY551928) and shown to be 5319 bp in size. Specific 

primers were developed for amplification of this 5319 bp fragment. PCR with these 

primers resulted in amplification of the 5319 bp fragment in all M. hyopneumoniae 

isolates tested. Sequencing of the amplicons obtained in high, moderate and low 

virulence isolates revealed only minor differences in the OPA-3 binding sites. The 

observed differences were not consistent with the virulence of the isolates. It remains 

unclear why RAPD with the OPA-3 primers did not result in a 5 kbp band in low 

virulence isolates. Possibly, some smaller bands were preferentially amplified in low 

virulence isolates compared to isolates of higher virulence, inhibiting amplification of 

the 5 kbp band during RAPD analyses. It can be concluded that although RAPD with 

OPA-3 might be useful to predict virulence of M. hyopneumoniae isolates, it is clear 

that the band itself does not carry any gene that may explain the differences in 

virulence of isolates observed in vivo. 

The fact that clear differences in virulence between isolates were observed 

may have important consequences at herd level. It is likely that herds infected with 

high virulence M. hyopneumoniae isolates will experience considerably higher 

economical losses than herds infected with low virulence isolates. A diagnostic test 

discriminating high and low virulence isolates would be very useful to implement the 

most appropriate control strategies. 

The availability of a collection of M. hyopneumoniae isolates of well 

determined virulence creates various possibilities for further research. Using our 

strains, Meyns et al. (2004) quantified the spread of high and low virulence isolates. 

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Although a faster spread of high virulence isolates was observed, the adjusted 

reproduction ratio (R n ) values were not significantly different. Macrophages are found 

to be the dominant cells present in lungs infected with M. hyopneumoniae (Sarradell 

et al., 2003) and pro-inflammatory cytokines which are mainly produced by 

macrophages play a prominent role in the lesion development (Henderson et al., 

1996). It would not be inconceivable that infections with high virulence isolates result 

in higher production of pro-inflammatory cytokines by macrophages compared to 

infections with low virulence isolates. A difference in macrophage stimulation was 

observed by Jungi et al. (1996) comparing different strains of Mycoplasma mycoides 

ssp. mycoides small colony type. In Mycoplasma fermentans, a lipoprotein called 

macrophage activating lipoprotein 2 kDa (MALP-2), was characterized and found to 

be responsible for the induction of pro-inflammatory cytokines (Seya and Matsumoto, 

2002). Further studies on lipoproteins present in M. hyopneumoniae might provide 

more insights in the pathogenesis of enzootic pneumonia. 

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The second part of this PhD deals with in vitro and in vivo susceptibility of M. 

hyopneumoniae isolates to some groups of antimicrobials. This information is 

essential to avoid ineffective treatments. Since M. hyopneumoniae is a very fastidious 

organism that is difficult to isolate and cultivate, data on the prevalence of acquired 

resistance are very scarce in the literature. Also, susceptibility information from other 

countries may be misleading since the type of antimicrobials and the amount 

administered may differ substantially due to country regulations in antimicrobial use, 

eg. fluoroquinolones are not allowed for use in pigs in the US but are allowed in the 

EU (World Health Organization, 1998; American Veterinarian Medical Association, 

2005). 

Based on the literature (Hannan et al., 1989; Ter Laak et al., 1991; Tanner et 

al., 1993; Friis and Szancer, 1994; Hannan et al., 1997), a high prevalence of isolates 

with acquired resistance was not expected. Only Inamoto et al. (1994) found several 

M. hyopneumoniae isolates with decreased susceptibility to oxytetracycline. In our 

studies, acquired resistance to tetracyclines was not detected in spite of the frequent 

use of these antibiotics in swine herds. One of the 21 Belgian M. hyopneumoniae 

isolates tested was found to have decreased susceptibility to macrolides and 

lincosamides, while in 5 isolates the susceptibility towards fluoroquinolones was 

decreased. Macrolides and lincosamides are frequently used for the treatment of M. 

hyopneumoniae infections in Western Europe, while fluoroquinolones are only 

occasionally used (Chauvin et al., 2002; Timmerman et al., 2005). 

Acquired resistance to an antimicrobial is the result of an alteration of the 

genome of a microorganism. It may arise because of a mutation in a gene or through 

horizontal transfer of resistance genes. Transfer-resistance has been described in M. 

pulmonis (Teachman et al., 2002) but no other reports on horizontal transfer of 

resistance genes exist. In chapter 2.2.2, it was shown that resistance to macrolides and 

lincosamides in our M. hyopneumoniae isolate was due to a A2058G mutation in the 

23S rRNA gene. A C264A mutation in parC and a C635T mutation in gyrA were 

found to be responsible for decreased susceptibility to enrofloxacin and flumequine 

(chapter 2.2.3). These mutations result in a modification of the target site for these 

antimicrobials. Resistance against fluoroquinolones might also be efflux mediated as 

has been described in M. hominis where reduced ciprofloxacin accumulation was 

related to the overexpression of two genes encoding a putative ABC type efflux 

(Raherison et al., 2002). However, it has been demonstrated that mutations in genes 

- 180 -


encoding the target sites of fluoroquinolones are the major cause of the increase in 

MIC and efflux mediated resistance is only supplementary (Poole, 2005). Whether 

efflux-mechanisms contribute to the increased MIC-values of fluoroquinolones for M. 

hyopneumoniae needs further investigation. 

Several factors influence the development and spread of antimicrobial 

resistant micro-organisms. The antimicrobial use is one factor gaining much attention. 

The selection pressure exerted by antimicrobials is influenced by the drug regimen, 

which includes the dose, the treatment interval, the duration of treatment and the 

formulation (Catry et al., 2003). A theoretical explanation of the influence of these 

factors on the development of de novo resistance is the “mutant selection window” 

(Catry et al., 2003; Drlica, 2003; Epstein et al., 2004). The mutant selection window 

is an antimicrobial concentration range extending from the MIC 99 to the mutant 

prevention concentration (MPC) which can be defined as the antimicrobial 

concentration required for blocking the growth of the least susceptible, single-step 

mutant. Antimicrobial concentrations fitted within this window enrich subpopulations 

selectively. Traditional dosing recommendations to exceed MICs are likely to place 

drug concentrations inside the selection window. In our study (chapter 2.2.1), the use 

of enrofloxacin in suckling piglets was striking. This drug was at least used in the 

farrowing unit on all herds where a resistant isolate was obtained. Delsol et al. (2004 

a;b) demonstrated that administration of registered doses of enrofloxacin for 5 days to 

weaned piglets selected for enrofloxacin resistant Campylobacter coli isolates and for 

quinolone (but not fluoroquinolone) resistant Salmonella enterica serovar 

Typhimurium isolates. The use of enrofloxacin in suckling piglets may have selected 

low level resistant strains due to a mutation in parC. This low level resistant 

population may have been sustained on the herd by the use of a drug dose within the 

mutant selection window possibly resulting in selection of a population with higher 

level resistance on one herd due to an additional mutation in gyrA. The same 

hypothesis could be raised concerning the use of lincomycin which on one herd may 

have resulted in spread of mutants in the 23S rRNA gene. Experimental studies 

designed to substantiate this hypothesis may provide practical evidence for future 

treatment regimens. 

In a final study, our experimental infection model was used to evaluate in vivo 

effectiveness of tylosin (chapter 2.2.4). It was shown that this antimicrobial, 

belonging to the 16-membered macrolides, can be used to limit the extent of 

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macroscopic lung lesions and consequently to reduce the clinical signs. Macrolide 

antibiotics accumulate in many tissues such as the epithelial lining fluid and it has 

been shown that the macrolide concentration in respiratory tract tissues and 

extracellular fluids is higher than simultaneously measured serum concentrations (Jain 

and Danzinger, 2004). Results of immunofluorescence studies demonstrated that 

although tylosin treatment is able to reduce the number of M. hyopneumoniae 

organisms in the lungs, not all bacteria are eliminated. The reason for the latter 

finding is not clear but narrowing of small blood vessels in the lungs due to 

accumulation of inflammatory cells around small airways and blood vessels may play 

a role. Diffusion of the drug from the blood to the lung tissue might be delayed or 

hampered resulting in diposition of the drug mainly at places with lower numbers of 

organisms. Additionally, the location of M. hyopneumoniae on the apex of the cilia 

and microvilli may also be a disadvantage for the accessiblity by the drug. 

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susceptibility of Mycoplasma hyopneumoniae isolated from swine. Journal of Veterinary 


17. Jain, R. & Danzinger, L.H. (2004). The macrolide antibiotics: a pharmacokinetic and 

pharmacodinamic overview. Current Pharmaceutical Design 10, 3045-3053. 

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27. Ro, L. & Ross, R.F. (1983). Comparison of Mycoplasma hyopneumoniae strains by serologic 

methods. American Journal of Veterinary Research 44, 2087-2094. 

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Allaire S.D., Taylor D.J (Editors), Diseases of Swine, 8th Edition, Iowa State University Press, 

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A., Lorenzo, H., Herráez, P. & Rodríguez, F. (2003). A morphological and 

immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally 

infected with Mycoplasma hyopneumoniae. Veterinary Pathology 40, 395-404. 

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(1991). Comparison of methods for in vitro testing of susceptibility of porcine Mycoplasma 


33. Teachman, A.M., French, C.T., Yu, H., Simmons, W.L. & Dybvig, K. (2002). Gene transfer 

in Mycoplasma pulmonis. Journal of Bacteriology 184, 947-951. 

34. Timmerman T., Dewulf J., Catry B., Feyen B., Opsomer G., de Kruif A. & Maes D. (2005). 

Quantification and evaluation of antimicrobial-drug use in group treatments for fattening pigs in 

Belgium. Preventive Veterinary Medicine, accepted. 

35. Wallgren, O., Bolske, G., Gustafsson, S., Mattsson, S., Fossum, C. (1998). Humoral immune 

responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of 

mycoplasmosis. Veterinary Microbiology 60, 193-205. 

36. World Health Organization. (1998). Use of quinolones in food animals and potential impact 

on human health. (Online). http:www.who.int/emcdocuments/zoonoses/docs/whoemczdi9810.html#Quinolone%20use%20in%animals. 

- 185 -

- 187 - 

4 SUMMARY

4 SUMMARY 

Mycoplasma hyopneumoniae is the primary cause of enzootic pneumonia in 

pigs, a chronic disease which is responsible for major economic losses in the pig 

industry worldwide. The mean factors influencing the disease-course and economic 

losses mentioned in the literature are environmental factors and herd management. 

A review of the literature focussing on the pathogenesis of enzootic 

pneumonia is given in chapter 1.1.1 of this PhD thesis. In chapter 1.1.2, an overview 

of antimicrobial agents and their in vitro and in vivo activity against M. 

hyopneumoniae is presented. Development of resistance in Mycoplasma spp. to 

circumvent the antibiotic action and the related resistance mechanisms are 

summarized. Control of enzootic pneumonia can be achieved using preventive 

medication or vaccination. Advantages and disadvantages of both strategies are 

discussed. Finally, M. hyopneumoniae eradication strategies developed and applied in 

some countries are described. 

The general aims of this thesis were to evaluate the virulence of M. 

hyopneumoniae field isolates and to study their antimicrobial susceptibility. 

In chapter 2.1.1, the patterns of M. hyopneumoniae infections were 

investigated in five herds with clinical evidence of enzootic pneumonia and in five 

herds subclinically infected with M. hyopneumoniae. In the clinically infected herds, 

housing and management conditions were good whereas these conditions were poor 

in the subclinically infected herds. In each herd, serum antibodies against M. 

hyopneumoniae were determined in pigs of different ages and nasal swabs were taken 

for M. hyopneumoniae detection using nested PCR (nPCR). The percentage of 

seropositive pigs in the clinically infected herds increased from 8% in pigs of 9 weeks 

to 52% in pigs of 18 weeks and seroconversion was most frequently observed 

between 12 and 15 weeks of age. In the subclinically infected herds, the percentages 

increased from 2 to 24% and most of the pigs became seropositive between 15 and 18 

weeks. The percentage of nPCR positive pigs at 6 weeks was 16% and 0% in the 

clinically and subclinically infected herds, respectively. The results demonstrate that 

the seroprevalences were higher in the clinically infected herds and that most of the 

pigs became infected with M. hyopneumoniae at a younger age. It can be concluded 

that additional factors different from housing and management, like differences in 

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4 SUMMARY 

virulence among M. hyopneumoniae isolates, may determine the infection pattern of 

M. hyopneumoniae and the clinical course of the infection. 

The next chapter (chapter 2.1.2) investigates the virulence of M. 

hyopneumoniae isolates under experimental conditions. These isolates were obtained 

from the herds selected as described in chapter 2.1.1. This study also aimed to link 

genetic characteristics, as determined by randomly amplified polymorphic DNA 

(RAPD), with the virulence of the isolates. Ninety conventional M. hyopneumoniaefree 

piglets were inoculated intratracheally with a field isolate, a reference strain of 

high virulence or sterile culture medium. The animals were examined daily for the 

presence of disease signs and a respiratory disease score (RDS) was assessed for 

every pig. Twenty-eight days post infection, pigs were euthanized, blood sampled and 

a lung lesion score was given per pig. Lung samples were processed for 

histopathology, immunofluorescence testing for M. hyopneumoniae and isolation of 

M. hyopneumoniae. RAPD analysis was performed on all M. hyopneumoniae isolates. 

Significant differences between isolates were found for the RDS, lung lesion 

score, histopathology, immunofluorescence and serology. Based on the results of the 

different parameters, isolates were divided into three “virulence” groups: low, 

moderate and high virulence isolates. Typically, a 5000 bp RAPD fragment was 

associated with the high and moderate virulence isolates whereas it was absent in low 

virulence isolates. It was concluded that high variation in virulence exists between M. 

hyopneumoniae isolates obtained from different pig herds and that further studies are 

required to determine whether the 5000 bp band obtained in the RAPD analysis can 

be used as a virulence marker. 

In the second part of this thesis, the susceptibility of M. hyopneumoniae to 

antimicrobial agents is described. Chapter 2.2.1 reports the in vitro susceptibilities of 

21 M. hyopneumoniae isolates obtained between 2000 and 2002. This study was 

conducted because information about in vitro susceptibilities of M. hyopneumoniae is 

scarce. A broth microdilution technique was used to determine the susceptibilities for 

12 antimicrobials frequently used in pig herds in Belgium. Acquired resistance to 

spectinomycin, oxytetracycline, doxycycline, gentamicin, florfenicol and tiamulin 

was not observed. One isolate showed acquired resistance to lincomycin, tilmicosin 

and tylosin. This isolate was susceptible to all other antibiotics tested. The Minimal 

Inhibitory Concentration (MIC)-values of flumequine were > 16 µg/ml for 5 isolates, 

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4 SUMMARY 

while the MIC 50 -value was 2 µg/ml. For these 5 isolates, the MIC-values for 

enrofloxacin were ≥ 0.5 µg/ml, the MIC 50 being 0.06 µg/ml. As far as we know, this 

is the first report of acquired resistance against macrolides, lincosamides and 

fluoroquinolones in M. hyopneumoniae field isolates. 

In the following two chapters (2.2.2 and 2.2.3), the mechanisms of acquired 

resistance of M. hyopneumoniae isolates against macrolides, lincosamides and 

streptogramins (MLS) and fluoroquinolones were elucidated. 

To study the acquired resistance to MLS antibiotics (chapter 2.2.2), the 

phenotype and the genotype of the resistant M. hyopneumoniae field isolate were 

compared to 5 susceptible isolates. Additional MICs were determined for other 

members of the MLS group, eg. erythromycin, clindamycin, azithromycin and 

clarithromycin. The MICs of all antibiotics tested were significantly higher for the 

resistant isolate. The MICs of the 16-membered macrolide tylosin ranged from 8 to 16 

µg/ml for the resistant isolate and from 0.03 to 0.125 µg/ml for the 5 susceptible 

isolates. The MICs for the 15-membered macrolides and lincosamides were higher 

than 64 µg/ml for the resistant isolate while only 0.06 to 0.5 µg/ml for the susceptible 

isolates. M. hyopneumoniae isolates are intrinsically resistant to the 14-membered 

macrolides due to a G2057A mutation (E. coli numbering) in their 23S rDNA. 

Therefore, high MICs were observed for all isolates, although the MICs for the 

resistant isolate were clearly increased. An additional, acquired A2058G point 

mutation was found in the 23S rRNA gene of the resistant isolate. No differences 

linked to resistance were found in the ribosomal proteins L4 and L22. The present 

study showed that 23S rRNA mutations, resulting in resistance to macrolides and 

lincosamides as described in other Mycoplasma spp., also occur under field conditions 

in M. hyopneumoniae. 

The mechanism of acquired resistance to fluoroquinolones in 5 M. 

hyopneumoniae field isolates was studied in chapter 2.2.3. Therefore, the genotypes 

of the 5 resistant isolates were compared to the genotypes of 5 susceptible isolates as 

determined in the study described in chapter 2.2.1. Their quinolone resistancedetermining 

regions (QRDR) of gyrA, gyrB, parC and parE were characterized. Parts 

of the DNA gyrase subunits, gyrA and gyrB, and the topoisomerase subunits, parC 

and parE, containing the QRDR, were sequenced. In all 5 resistant isolates, one point 

mutation (C → A) in parC was found, resulting in an amino acid change from serine 

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4 SUMMARY 

to tyrosine at position 80 (E. coli numbering). For 4 of these isolates, this was the only 

mutation found. These isolates had a MIC of enrofloxacin of 0.5 µg/ml, while for 

sensitive isolates the MIC of enrofloxacin was ≤ 0.06 µg/ml. One resistant isolate (Mh 

20) had an extra mutation (C → T) in gyrA resulting in an amino acid change from 

alanine to valine at position 83 (E. coli numbering). This mutation was correlated with 

an increase in the MIC of enrofloxacin (> 1 µg/ml). No mutations resulting in an 

amino acid change were detected in the QRDR of the gyrB and parE genes of the 

selected stains. This is the first description of the mechanism of resistance against 

fluoroquinolones in M. hyopneumoniae. 

In the last chapter (chapter 2.2.4), the efficacy of in-feed medication with 

tylosin (Tylan® Premix; Elanco Animal Health) for the treatment of enzootic 

pneumonia was examined in an experimental M. hyopneumoniae infection model. 

Three groups of 10 conventional M. hyopneumoniae-free piglets were inoculated 

intratracheally with a highly virulent M. hyopneumoniae field isolate (non-treated 

control (non-TP) group and tylosin premix (TP group)) or with sterile culture medium 

(non-infected and non-treated control (NC) group). Twelve days after infection when 

10% of the animals were coughing, tylosin treatment of the TP group was initiated 

(100 mg/kg of feed for 21 days). Animals were daily examined for the presence of 

clinical signs and a respiratory disease score (RDS) was given per pig. Thirty-three 

days after infection, all pigs were euthanized and the lung lesions were quantified. 

Lung samples were processed for immunofluorescence testing for M. hyopneumoniae. 

Average RDS and lung lesion scores were significantly higher (P

4 SUMMARY 

In conclusion, the most important findings of this research are that: 

- apart from management and housing conditions, the virulence of the M. 

hyopneumoniae isolate strongly influences the disease-course 

- antimicrobial resistance is not a major problem in M. hyopneumoniae isolates 

but it does occur in the field and careful use of antimicrobial agents is 

warranted 

- the mechanism of resistance in M. hyopneumoniae against MLS and 

fluoroquinolones is based on mutations in genes encoding the drug target site 

- tylosin (100 ppm) administered in the feed 12 days after experimental 

infection is efficient in reducing clinical signs and lung lesions 

These findings are generally discussed in chapter 3 and possibilities for future 

research are also indicated. 

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- 195 - 

5 SAMENVATTING

5 SAMENVATTING 

Mycoplasma hyopneumoniae is de primaire oorzaak van enzoötische 

pneumonie bij varkens, een chronische ziekte die wereldwijd verantwoordelijk is voor 

grote economische verliezen in de intensieve varkenshouderij. In de literatuur wordt 

vermeld dat het ziekteverloop en de economische verliezen voornamelijk beïnvloed 

worden door de omgevingsfactoren en het bedrijfsmanagement. 

In hoofdstuk 1.1.2 wordt een overzicht gegeven van de beschikbare 

antimicrobiële middelen en hun in vivo en in vitro activiteit tegen M. hyopneumoniae. 

Ontwikkeling van antimicrobiële resistentie door Mycoplasma spp. en de gerelateerde 

resistentiemechanismen worden samengevat. Voor het bestrijden van enzoötische 

pneumonie worden in de praktijk zowel preventieve medicatie als vaccinatie gebruikt. 

De voordelen en nadelen van beide strategieën worden bediscussieerd. Tot slot 

worden eradicatiestrategieën voor M. hyopneumoniae, die ontwikkeld en toegepast 

werden in een aantal landen, besproken. 

De voornaamste doelstellingen van deze doctoraatsthesis waren het evalueren 

van de virulentie van M. hyopneumoniae veldisolaten en het bestuderen van hun 

antimicrobiële gevoeligheid. 

In hoofdstuk 2.1.1 werden de infectiepatronen van M. hyopneumoniae 

onderzocht op 5 bedrijven met een klinisch beeld van enzoötische pneumonie en op 5 

subklinisch geïnfecteerde bedrijven. Op de klinisch geïnfecteerde bedrijven waren de 

huisvesting en het management goed terwijl deze onvoldoende waren op de 

subklinisch geïnfecteerde bedrijven. Op elk bedrijf werd van een aantal 

leeftijdscategorieën bloed genomen voor het opsporen van antistoffen tegen M. 

hyopneumoniae en werden neusswabs verzameld waarin de kiem werd opgezocht 

d.m.v. nested PCR (nPCR). Het percentage seropositieve dieren op klinisch 

geïnfecteerde bedrijven steeg van 8% bij biggen van 9 weken oud tot 52% bij varkens 

van 18 weken. De meeste dieren seroconverteerden tussen 12 en 15 weken leeftijd. 

Op de subklinisch geïnfecteerde bedrijven stegen de percentages van 2 naar 24%. De 

meeste dieren seroconverteerden tussen 15 en 18 weken. Het percentage nPCR 

positieve biggen van 6 weken was 16% op de klinisch geïnfecteerde bedrijven en 0% 

op de subklinisch geïnfecteerde bedrijven. Deze resultaten tonen aan dat de 

seroprevalenties hoger zijn op klinisch geïnfecteerde bedrijven en dat op deze 

bedrijven de meeste biggen geïnfecteerd worden op een jongere leeftijd. Hieruit 

- 197 -


kunnen we concluderen dat niet alleen de huisvesting en het management invloed 

hebben op het infectiepatroon van M. hyopneumoniae en op het klinisch verloop van 

de ziekte. Mogelijks bestaan er ook verschillen in virulentie tussen M. hyopneumoniae 

isolaten. 

In het volgende hoofdstuk (hoofdstuk 2.1.2) werd de virulentie van zes M. 

hyopneumoniae veldisolaten onderzocht onder experimentele omstandigheden. Deze 

isolaten werden bekomen op de bedrijven beschreven in hoofdstuk 2.1.1. In deze 

studie werd ook het verband nagegaan tussen genetische verschillen van de isolaten, 

bepaald met behulp van randomly amplified polymorphic DNA (RAPD), en de 

virulentie van de isolaten. Negentig conventionele M. hyopneumoniae-vrije biggen 

werden intratracheaal geïnoculeerd met een veldisolaat, een hoog-virulente 

referentiestam of steriel cultuurmedium. Dagelijks werden de klinische symptomen 

van de dieren beoordeeld door middel van een “respiratory disease score” (RDS). 

Achtentwintig dagen na de infectie werden alle biggen geëuthanaseerd, werd er bloed 

genomen en werden de longlesies per varken gescoord. Longstalen werden verwerkt 

voor histopathologisch onderzoek, immunofluorescentie en isolatie van M. 

hyopneumoniae. RAPD analyse werd uitgevoerd op alle M. hyopneumoniae isolaten. 

Significante verschillen tussen de isolaten werden waargenomen voor de RDS, de 

longlesies, de histopathologie, de immunofluorescentie en de serologie. Gebaseerd op 

de resultaten van deze parameters werden de isolaten in 3 groepen verdeeld: hoog-, 

matig- en laagvirulente isolaten. Opvallend was de aanwezigheid van een 5000 bp 

RAPD fragment bij alle hoog- en matig-virulente isolaten. Dit fragment was afwezig 

bij laagvirulente isolaten. Uit dit onderzoek kon besloten worden dat er een grote 

variatie is in de virulentie van M. hyopneumoniae isolaten die geïsoleerd werden op 

de verschillende bedrijven. Bijkomende studies zijn nodig om de 5000 bp band verder 

te karakteriseren en om na te gaan of deze kan gebruikt worden als virulentiemerker. 

In het tweede deel van deze thesis werd ingegaan op de gevoeligheid van M. 

hyopneumoniae tegen antimicrobiële agentia. In hoofdstuk 2.2.1 werd de gevoeligheid 

bepaald van 21 M. hyopneumoniae isolaten, die verzameld werden tussen 2000 en 

2002, voor 12 antibiotica die in de Belgische varkenshouderij frequent worden 

gebruikt. Hierbij werd gebruik gemaakt van een bouillon microdilutie techniek. 

Verworven resistentie tegen spectinomycine, oxytetracycline, doxycycline, 

gentamicine, florfenicol en tiamulin werd niet waargenomen. Eén isolaat was resistent 

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tegen lincomycine, tilmicosine en tylosine, maar gevoelig voor alle andere geteste 

antibiotica. De Minimale Inhibitorische Concentratie (MIC)-waarden van flumequine 

waren > 16 µg/ml voor 5 isolaten, terwijl de MIC 50 2 µg/ml bedroeg. De MICwaarden 

voor enrofloxacine waren ≥ 0,5 µg/ml voor deze 5 isolaten, terwijl de MIC 50 

0,06 µg/ml bedroeg. Dit is de eerste melding van verworven resistentie tegen 

macroliden, lincosamiden en fluoroquinonoles voor M. hyopneumoniae isolaten. 

In de volgende twee hoofdstukken (2.2.2 en 2.2.3) werden de mechanismen 

die verantwoordelijk zijn voor deze verworven resistentie bepaald. 

Voor het bestuderen van de verworven resistentie tegen macroliden en 

lincosamiden (hoofdstuk 2.2.2) werden het fenotype en het genotype van de resistente 

M. hyopneumoniae stam vergeleken met 5 gevoelige isolaten. MICs werden bepaald 

voor de 14-ring macroliden erythromycine en clarythromycine, het 15-ring macrolide 

azithromycine, het 16-ring macrolide tylosine en de lincosamiden lincomycine en 

clindamycine. De MIC was voor alle antibiotica significant hoger voor de resistente 

stam. De MICs van tylosine varieerden van 8 tot 16 µg/ml voor de resistente stam en 

van 0,03 tot 0,125 µg/ml voor de 5 gevoelige isolaten. De MICs van azithromycine en 

de lincosamiden waren hoger dan 64 µg/ml voor de resistente stam, maar bedroegen 

slechts 0,06 tot 0,5 µg/ml voor de gevoelige isolaten. M. hyopneumoniae isolaten zijn 

intrinsiek resistent tegen 14-ring macroliden omwille van een G2057A transitie (E. 

coli nummering) in hun 23S rDNA. De MICs van erythromycine waren voor alle 

isolaten 8-32 µg/ml. Voor de resistente stam was de MIC nog duidelijk hoger dan 

voor de andere isolaten, namelijk >64 µg/ml. Een bijkomende, verworven A2058G 

puntmutatie werd gevonden in het 23S rRNA gen van de resistente stam. In de 

ribosomale proteïnes L4 en L22 werden geen verschillen teruggevonden die gelinkt 

konden worden aan resistentie. Dit onderzoek toont aan dat een mutatie in het 23S 

rRNA resulteert in resistentie tegen macroliden en lincosamiden zoals eerder 

beschreven voor andere Mycoplasma spp. en dat deze mutatie voorkomt onder 

praktijkomstandigheden. 

Het mechanisme voor verworven resistentie van 5 M. hyopneumoniae isolaten 

tegen fluoroquinolones werd verder bestudeerd in hoofdstuk 2.2.3. Hiertoe werd het 

genotype van de 5 resistente isolaten vergeleken met het genotype van 5 gevoelige 

isolaten. De quinolone resistentie bepalende regio’s (“Quinolone resistancedetermining 

region: QRDR) van de genen gyrA, gyrB, parC en parE werden 

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gekarakteriseerd. Hiervoor werden delen van de DNA gyrase subunits, gyrA en gyrB, 

en de topoisomerase subunits, parC en parE, die de QRDR bevatten, gesequeneerd. In 

de 5 resistente isolaten werd een puntmutatie (C → A) in parC gevonden. De 

puntmutatie resulteerde in een aminozuurwijziging van serine naar tyrosine op positie 

80 (E. coli nummering). Voor 4 van deze isolaten was dit de enige mutatie die 

gevonden werd. De MIC van enrofloxacine voor deze isolaten was 0,5 µg/ml terwijl 

de MIC van de gevoelige isolaten ≤ 0,06 µg/ml bedroeg. Eén resistente stam met een 

MIC van >1 µg/ml voor enrofloxacine had een extra mutatie (C → T) in gyrA die 

resulteerde in een aminozuurwijziging van alanine naar valine op positie 83 (E. coli 

nummering). Mutaties die resulteren in een aminozuurwijziging werden niet 

waargenomen in de QRDR van de gyrB en parE genen van de geselecteerde isolaten. 

Dit onderzoek beschrijft voor het eerst het mechanisme van fluoroquinoloneresistentie 

van M. hyopneumoniae isolaten. 

In het laatste hoofdstuk (hoofdstuk 2.2.4) werd het onderzoek beschreven naar 

het effect van voedermedicatie met tylosine (Tylan® Premix; Elanco Animal Health) 

op het verloop van een experimentele M. hyopneumoniae infectie. Drie groepen van 

10 conventionele M. hyopneumoniae-vrije biggen werden intratracheaal geïnoculeerd 

met een hoogvirulente M. hyopneumoniae stam die in vitro gevoelig was voor 

tylosine (niet-behandelde controle groep (non-TP) en tylosine premix groep (TP)) of 

met steriel cultuurmedium (niet-geïnfecteerde en niet-behandelde controle (NC) 

groep). Twaalf dagen na de infectie, op het moment dat 10% van die dieren hoestte, 

werd de behandeling met tylosine gestart (100 mg/kg voeder gedurende 21 dagen). De 

dieren werden dagelijks gecontroleerd op de aanwezigheid van klinische symptomen 

en aan iedere big werd een “respiratory disease score” (RDS) gegeven. Drieëndertig 

dagen na inoculatie werden alle biggen geëuthanaseerd en werden de longen 

onderzocht op de aanwezigheid van letsels. De gemiddelde RDS en longlesiescores 

waren significant hoger in de beide geïnfecteerde groepen vergeleken met de NC 

groep. De gemiddelde RDS op 23 tot 33 dagen na infectie was significant hoger 

(P


De belangrijkste bevindingen van dit doctoraatsonderzoek zijn dat: 

- behalve het management en de huisvesting ook de virulentie van de M. 

hyopneumoniae stam een sterke invloed heeft op het ziekteverloop van 

enzoötische pneumonie 

- antibioticumresistentie nog geen groot probleem vormt voor M. 

hyopneumoniae isolaten maar wel aanwezig is. Daarom is voorzichtig 

omspringen met antimicrobiële middelen noodzakelijk 

- het mechanisme voor verworven resistentie van M. hyopneumoniae isolaten 

tegen macroliden - lincosamiden en fluoroquinolones berust op mutaties in de 

genen die coderen voor de doelwitplaats van deze antimicrobiële middelen 

- voerdermedicatie met tylosine (100 ppm) de klinische symptomen en de 

longlesies te wijten aan een M. hyopneumoniae infectie, efficiënt reduceert. 

Deze bevindingen worden verder bediscussieerd in hoofdstuk 3 en 

mogelijkheden voor verder onderzoek worden aangegeven. 

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DANKWOORD

DANKWOORD 

Het heeft behoorlijk wat moeite gekost om dit werk te voltooien en op sommige 

momenten was een stevige dosis koppigheid hard nodig. Maar nu het zover is, ben ik 

bijzonder blij dat ik toch heb volgehouden, want een doctoraat opent wegen waarvan 

ik anders maar zou kunnen dromen. 

Het is onmogelijk om een doctoraat op je eentje te maken. De vele mensen die hierbij 

een groot of klein handje hebben bijgedragen wil ik dan ook uitdrukkelijk bedanken. 

Allereerst zijn er mijn promotoren, Prof. Haesebrouck en Prof. Maes. Prof. 

Haesebrouck, alhoewel ik tijdens de eerste jaren met een klein hartje bij u langs kwam 

om door u verbeterde teksten op te halen, heb ik deze verbeteringen en opmerkingen 

steeds meer weten te appreciëren. Ze bleken steeds correct en terecht. Soms vraag ik 

me af waar u de moed en tijd vandaan haalt om telkens weer teksten van verschillende 

personen zo nauwgezet te lezen en verbeteren. Daarnaast waardeer ik uw inzicht in en 

realistische kijk op het onderzoek, een absolute noodzaak om de experimenten 

relevant en uitvoerbaar te houden. Prof. Maes, Dominiek, jij was de persoon bij wie ik 

al eens sneller binnen liep met allerhande vragen. Samen met Nathalie en Tim zijn we 

de eerste doctorandi met u als promotor, en we verdedigen ons doctoraat dan nog in 

dezelfde maand. Dominiek, bedankt voor het discussiëren, meedenken, lezen van 

teksten,.... 

Prof. de Kruif, bedankt dat u mij de kans hebt gegeven om aan uw vakgroep te 

werken. De vrijheid die u uw assistenten geeft, is zeer stimulerend! Ondanks uw 

drukke agenda vond u toch steeds de tijd om interesse te tonen voor mijn onderzoek 

en de artikels en ten slotte om dit doctoraat na te lezen. Bedankt ook Prof. em. 

Verdonck, Marc, als bezieler van de varkenssektor in deze vakgroep en voor je 

interesse in het varkensonderzoek. 

Bedankt ook leden van de begeleidings- en examencommissie (Prof. J. Mainil, Prof. 

P. Deprez, Prof. P. De Backer, Dr. P. Butaye, Prof. H. Nauwynck en Prof. P. 

Wallgren) voor het kritisch lezen van dit doctoraat. Uw opmerkingen droegen zeker 

bij tot de kwaliteit ervan. 

Dit doctoraat was niet mogelijk zonder de financiële steun van de FOD 

Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu. Ik wil in het 

bijzonder Dr. X. Van Huffel en Ir. J. Weerts bedanken voor hun vertrouwen in dit 

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DANKWOORD 

onderzoek en voor de stimulerende opmerkingen tijdens de begeleiding van dit 

project. 

Voor dit onderzoek was er een nauwe samenwerking met het CODA. Deze 

samenwerking werd mogelijk gemaakt door Dr. J. Peeters. Mijn eerste collega in het 

CODA was Lies Thermote. Lies, het was heel aangenaam om met u samen te werken. 

We voelden elkaar goed aan. Het speet me erg toen je vertelde dat je van werk ging 

veranderen, maar ik begreep je redenen wel. Gelukkig hebben we contact gehouden. 

Het is spijtig dat je er vandaag niet kan zijn (het is wat moeilijk om snel even langs te 

komen vanuit Vietnam) want jou bijdrage in dit mycoplasma onderzoek is niet gering, 

jij bent er immers in geslaagd de eerste M. hyopneumoniae stammen in België te 

isoleren. Inge, jij hebt mee dit project uit de grond gestampt en me in de beginfase 

mijn weg helpen vinden in de mycoplasma diagnostiek, bedankt. Ook Katrien, en 

daarna Saar wil ik bedanken voor hun technische assistentie, vooral voor het 

aanmaken van liters NHS20 medium. Tim, jij nam het werk van Lies over en plaatste 

het op een sneltrein, alhoewel je eerste ervaringen met het mycoplasma onderzoek 

behoorlijk frustrerend waren (5kb...), hoe kan het ook anders als je met M. 

hyopneumoniae werkt. Maar je bent er door je dynamiek toch in geslaagd op korte tijd 

een mooi doctoraat klaar te krijgen. Ik hoop dat je je weg in het onderzoek kan verder 

zetten! Patrick, bedankt om mij wegwijs te maken in het in vitro antibiotica 

onderzoek, uw stokpaardje. En nogmaals bedankt voor de tijd die je willen spenderen 

hebt in het lezen en bespreken van dit doctoraat. 

Een bijzonder woordje van dank wil ik ook richten aan alle varkenshouders die 

meegewerkt hebben aan dit onderzoek. Die ons toelieten om stalen te nemen op hun 

bedrijven, stalen die noodzakelijk waren voor de isolatie van M. hyopneumoniae, de 

basis van dit onderzoek. Ook de familie Malfait ben ik bijzonder dankbaar omdat zij 

steeds bereid waren om ons M. hyopneumoniae-vrije biggen te leveren, noodzakelijk 

voor de experimentele studies. Daarnaast waren hun slachtvarkens de leveranciers van 

bloed, nodig voor het bereiden van medium voor de isolatie van M. hyopneumoniae. 

Omdat ik zowel aan de vakgroep Voortplanting, Verloskunde en 

Bedrijfsdiergeneeskunde als de vakgroep Pathologie, Bacteriologie en 

Pluimveeziekten werkte, had ik veel collega’s. Te veel om hier op te noemen, met het 

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DANKWOORD 

risico dat ik bepaalde personen zou vergeten. Bedankt collega’s om er te zijn, voor de 

steun, voor de hulp, voor het plezier dat we samen hadden, voor het ondergaan van 

lastige buien,... En.... badmintonners en lopers, hou vol! Een gezonde geest in een 

gezond lichaam zeggen ze toch altijd, hier zit veel waarheid in. Blijf fruit eten.... 

Een extra woordje van dank aan het ATP van beide vakgroepen, jullie zijn een 

onmisbare hulp geweest voor mij. 

Marc Coryn, bedankt voor het zeer nauwgezet lezen van dit doctoraat en het 

uitfilteren van typ- en spellingsfouten. 

Nathalie, met jou heb ik een “samen uit, samen thuis” gevoel. We begonnen samen 

aan onze opleiding diergeneeskunde en erna aan ons doctoraat en we verdedigen het 

in dezelfde maand. Vanaf nu gaan we andere richtingen uit. Veel succes en plezier, 

geniet van je baby! Ook bedankt om gedurende 6 jaar mijn bureaugenootje te zijn. 

Tom en Dries, jullie gaan verder met het mycoplasma onderzoek. Veel succes ermee 

en laat je niet teveel frustreren. 

Sommige aspecten van het werk waren in tegenstelling tot wie ik ben. Zo is uren aan 

een computer of flow blijven zitten niet echt aan mij besteed. De proefvarkens voeren, 

hokken kuisen en eikels voor ze rapen in het bos, om ze toch maar een zo aangenaam 

mogelijk leven te geven, ging mij stukken beter af. Maar dan moet je weer afscheid 

nemen van dieren, intelligenter dan honden, waarmee je ondertussen een band hebt 

opgebouwd. Het werken met Mycoplasma hyopneumoniae vereiste immers 

langdurige proeven van minstens 5 weken. Toen ik op het einde van de eerste proef de 

varkens moest euthanaseren en hierdoor nogal ongelukkig rondliep was de 

boodschap: “de eerste keer voel je je slecht als je je proefdieren moet doden, maar het 

went en de 2 de keer gaat het al veel makkelijker”. Bij mij wende dat nooit. 

Dirk Lips en Chris De Pauw, ik ben jullie ongelooflijk dankbaar dat jullie me de kans 

geven om te werken aan de hogeschool KaHo St.-Lieven in St. Niklaas. Geert 

(Hoflack), jij hebt hier ook een belangrijke rol gespeeld, bedankt! Een reden waarom 

ik op mijn tanden gebeten heb en dit doctoraat heb afgewerkt, is omdat ik inzag dat 

het hebben van een doctoraat een noodzaak zou worden om te kunnen lesgeven aan 

een hogeschool. Ik kijk er naar uit om bij jullie te beginnen en hoop dat ik aan jullie 

verwachtingen zal kunnen voldoen. 

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DANKWOORD 

Siegrid, Gwen, Kaat, Ilse, Liesbet, jullie kunnen hier vandaag niet zijn omwille van 

piepjonge babies (van een babyboom gesproken!). Nu wij nog hé An. “Meiden van 

Gent”, ondertussen ben ik nog de enige Gentse, ik hoop dat we er toch nog in slagen 

gezellige avondjes en misschien af en toe een weekendje te organiseren. Ik zie jullie 

graag. 

Lieve, je bent een bijzondere vrouw. Ik heb veel respect voor je! 

Cathy en Griet, jullie zijn van onschatbare waarde voor mij. Ik hoop dat jullie dat 

beseffen! 

Jef, we hebben elkaar dankzij het werk leren kennen en zijn ondertussen vrienden 

geworden. Je was een ongelooflijke steun voor mij, ik ben blij dat ik je ken. 

Marc en Nicole, we moeten Maarten missen, maar gelukkig hebben we elkaar. 

Bedankt voor jullie nimmer aflatende interesse en steun. 

Joris, het is dan uiteindelijk toch niet gelukt tussen ons. Een moeilijke beslissing in 

een moeilijke periode. Maar het was de moeite waard, een mens groeit in zijn relaties 

en in die met jou ben ik veel gegroeid. 

En dan mama en papa en Nele en Ward, wat zou ik zijn zonder jullie Jullie merken 

het wel hé aan de frequentie dat ik thuis verschijn dat ik niet zonder jullie kan. 

Trouwens, thuis is nog steeds Kapellen. Gent is mijn huis. 

Jo 

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CURRICULUM VITAE

CURRICULUM VITAE 

Jo Vicca werd geboren op 31 december 1974 te Tienen. In 1992 beëindigde zij 

haar secundaire opleiding aan het Sint-Jozefsinstituut te Tienen in de richting 

Wetenschappelijke B. Na 1 jaar de opleiding graduaat chemie te hebben gevolgd aan 

de Katholieke Hogeschool Limburg, begon zij in 1993 aan de opleiding tot dierenarts 

aan de Faculteit Diergeneeskunde van de Universiteit Gent. Het diploma dierenarts 

werd in 1999 behaald met grote onderscheiding. Hierna was zij werkzaam als 

wetenschappelijk medewerker in het kader van een onderzoeksproject van de FOD 

Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu. Dit project dat 

handelde over “de studie van diversiteit van Mycoplasma hyopneumoniae in het kader 

van de bestrijding van enzoötische pneumonie” en werd uitgevoerd in samenwerking 

met de vakgroepen voortplanting, verloskunde en bedrijfsdiergeneeskunde en 

pathologie, bacteriologie en pluimveeziekten van de Faculteit Diergeneeskunde, 

Universiteit Gent en het Centrum voor Onderzoek in Diergeneeskunde en 

Agrochemie. Het onderzoek uitgevoerd tijdens dit project heeft geleid tot deze 

doctoraatsthesis. Jo Vicca is auteur en medeauteur van meerdere wetenschappelijke 

publicaties, nam deel aan verschillende wetenschappelijke congressen en gaf 

verschillende voordrachten aan dierenartsen en andere onderzoekers. 

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PUBLICATIONS

ARTICLES 

• Vicca, J., Maes, D.,Thermote, L., Peeters, J.,Haesebrouck, F. & de Kruif, A. 

(2002). Patterns of Mycoplasma hyopneumoniae infections in Belgian Farrowto-Finish 

pig herds with diverging disease course. Journal of Veterinary 


• Maes, D.,Verbeke, W.,Vicca, J.,Verdonck, M. & de Kruif, A. (2003). Benefit to 

cost of vaccination against Mycoplasma hyopneumoniae in pig herds under 

Belgian market conditions from 1996 to 2000. Livestock Production Science 

83, 85-93. 

• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A. & 

Haesebrouck, F. (2003). Evaluation of virulence of Belgian Mycoplasma 

hyopneumoniae field isolates. Veterinary Microbiology. 97, 177-190. 

• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., de Kruif, A. & Haesebrouck, F. 

(2004). In vitro susceptibilities of Mycoplasma hyopneumoniae field isolates. 


• Meyns, T., Maes, D., Dewulf, J., Vicca, J., Haesebrouck, F. & de Kruif, A. 

(2004). Quantification of the spread of Mycoplasma hyopneumoniae in 

nursery pigs using transmission experiments. Preventive Veterinary Medicine 

66, 265-275. 

• Vicca, J., Maes, D., Jonker, L., de Kruif, A. & Haesebrouck, F. (2005). Efficacy 

of in-feed medication with tylosin for the treatment and control of 

Mycoplasma hyopneumoniae infections. The Veterinary Record 156, 606-610. 

• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., De Baere, T., Verhelst, R., 

Peeters, J., de Kruif, A., Haesebrouck, F. & Vaneechoutte, M. (2005). 

Evaluation of amplified rDNA restriction analysis (ARDRA) for the 

identification of Mycoplasma species.BMC Infectious Diseases 5, 46. 

• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Peeters, J., de Kruif, A. & 

Haesebrouck, F. (2005). The diversity of Mycoplasma hyopneumoniae within 

and between herds using Pulsed-Field Gel Electrophoresis. Veterinary 


• Stakenborg, T., Vicca, J., Butaye, P., Imberechts, H., Peeters, J., de Kruif, A., 

Haesebrouck, F. & Maes, D. (2005). A multiplex PCR to identify porcine 

- 215 -

mycoplasmas present in broth cultures. Veterinary Research Communications, 

In Press. 

• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., Minion, F.C., Peeters, J., de 

Kruif, A. & Haesebrouck, F. (2005). Characterization of in vivo acquired 

resistance of Mycoplasma hyopneumoniae to macrolides and lincosamides. 

Microbial Drug Resistance, 11, 291-295. 

• Stakenborg, T., Vicca, J., Verhelst, R., Butaye, P., Maes, D., Naessens, A., 

Claeys, G., De Ganck, C., Haesebrouck, F. & Vaneechoutte, M. (2005). 

Evaluation of tDNA-PCR for the identification of Mollicutes. Journal of 

Clinical Microbiology, 43, 4558-4566. 

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POSTERS ON NATIONAL AND INTERNATIONAL CONGRESSES 

• Vicca, J., Maes, D., Thermote, L., Peeters, J., Haesebrouck, F. & de Kruif, A. 

Patterns of Mycoplasma hyopneumoniae infection in Belgian farrow-to-finish 

pig herds. 17 th Congress of the International Pig Veterinary Society, Ames, 

Iowa, USA, 02-05 June 2002. 

• Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. 

Haesebrouck. Evaluation of virulence of Belgian Mycoplasma hyopneumoniae 

Field Isolates. 17 th Congress of the International Pig Veterinary Society; 

Ames, Iowa, USA, 02-05 June 2002. 


Haesebrouck, F. Evaluation of virulence of Belgian Mycoplasma 

hyopneumoniae field isolates. 14th international congress of the International 

Organisation for Mycoplasmology; Vienna, Austria, 07-12 July 2002. 

• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., Peeters, J., de Kruif, A., 

Devriese, L. & Haesebrouck, F. Antibiotic susceptibility of Belgian 

Mycoplasma hyopneumoniae Field Isolates. 14th international congress of the 

International Organisation for Mycoplasmology; Vienna, Austria, 07-12 July 

2002. 

• Stakenborg, T., Vicca, J., Butaye, P., Maes, D., de Kruif, A. & Haesebrouck, F. 

RAPD analysis of Belgian Mycoplasma hyopneumoniae strains. 14th 

international congress of the International Organisation for Mycoplasmology; 

Vienna, Austria, 07-12 July 2002. 


Development of a multiplex PCR to detect Mycoplasmas present in the lungs 

of pigs. 14th international congress of the International Organisation for 

Mycoplasmology; Vienna, Austria, 07-12 July 2002. 

• Vicca, J., Stakenborg, T., Maes, D., Butaye, P., de Kruif, A. & Haesebrouck, F. 

Antimicrobial susceptibilities of Mycoplasma hyopneumoniae field isolates. 

11 th annual meeting of the Flemish society for veterinary epidemiology and 

economics; Torhout, Belgium, 11 December 2003. 


Haesebrouck, F. In vitro susceptibility of Mycoplasma hyopneumoniae field 

- 217 -

isolates. 18 th Congress of the International Pig Veterinary Society; Hamburg, 

Germany, 27 June-1 July 2004. 

• Meyns, T., Maes, D., Vicca, J., Dewulf, J., Haesebrouck, F. & de Kruif, A. Use 

of transmission experiments to quantify the spread of Mycoplasma 

hyopneumoniae in nursery pigs. 18 th Congress of the International Pig 

Veterinary Society, Hamburg, Germany, 27 June-1 July 2004. 


Optimisation of a Pulsed Field Gel Electrophoresis (PFGE) technique for 

Mycoplasma hyopneumoniae. 15th international congress of the International 

Organisation for Mycoplasmology; Athens, Georgia, USA, 11-16 July 2004. 


Characterization of a Mycoplasma hyopneumoniae field isolate resistant to 

MLS antibiotics. 15th international congress of the International Organisation 

for Mycoplasmology; Athens, Georgia, USA, 11-16 July 2004. 


Amplified Fragment Length Polymorphism (AFLP) of three porcine 

Mycoplasma spp. 15th international congress of the International Organisation 

for Mycoplasmology; Athens, Georgia, USA, 11-16 July 2004. 

• Stakenborg, T., Vicca, J., Verhelst, R., Butaye, P., Maes, D., Naessens, A., 

Clays, G., De Ganck, C., Haesebrouck, F. & Vaneechoutte, M.. Evaluation of 

tDNA-PCR for the identification of Mollicutes. Belgian Society of 

Microbiology, Brussels, 3 rd December 2004. 

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ORAL PRESENTATIONS 

• Vicca, J. Mycoplasma hyopneumoniae infectiepatronen in gesloten 

varkensbedrijven, gebruik makende van serologie en nested PCR op 

neusswabs. Symposium: Mycoplasma hyopneumoniae, inzichten in 

epidemiologie en immunologie, georganiseerd door Groep Geneeskunde van 

het Varken en Pfizer Animal Health, Ede, Nederland, 20 December 2000. 

• Vicca, J. Evaluation of virulence of Belgian Mycoplasma hyopneumoniae field 

isolates. 17 th Congress of the International Pig Veterinary Society, Ames, 

Iowa, USA, 02-05 June 2002. 

• Vicca, J. Onderzoek naar verschillen tussen Belgische Mycoplasma 

hyopneumoniae veldisolaten. International Pig Veterinary Society, Belgian 

branch studienamiddag, Faculteit Diergeneeskunde, Merelbeke, 22 november 

2002. 

• Vicca J. La broncho-pneumonie enzootique. Les voor 2de proef, 

longaandoeningen bij varkens, Faculté de Médécine Vétérinaire, Sart-Tilman, 

10 December 2002. 

• Vicca, J. Ronde tafel gesprekken Boehringer Ingelheim: infectiepatronen van 

Mycoplasma hyopneumoniae. 9x June 2003. 

• Vicca, J. Evaluation of virulence of Belgian Mycoplasma hyopneumoniae field 

isolates. Swine and wine (vereniging varkensdierenartsen), Roermond, 

Nederland, 27 September 2003. 

• Vicca, J. Virulentieverschillen en vroege spreiding van M. hyopneumoniae. Post 

Academisch Onderwijs in de Diergeneeskunde (PAOD), Houten, Nederland, 1 

December 2004. 

• Vicca, J. Infecties met Mycoplasma hyopneumoniae. Vakdierenarts varken, 

Faculteit Diergeneeskunde, UGent, 8 december 2004. 



december 2004. 



december 2004. 

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