identification of mollicutes and characterisation of...

GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Department of Pathology, Bacteriology, and Poultry Diseases

Department of Reproduction, Obstetrics, and Herd Health

Identification of mollicutes and characterisation

of Mycoplasma hyopneumoniae isolates

Tim STAKENBORG

Dissertation for the degree of Doctor of Veterinary Science (PhD)

at the Faculty of Veterinary Medicine, University of Ghent

2005

Promoters: Prof. Dr. F. Haesebrouck & Prof. Dr. D. Maes

Co-promoter: Dr. P. Butaye

the more you see, the less you know

(from the ‘City of Blinding Lights’ by U2)

TABLE OF CONTENTS

TABLE OF CONTENTS..................................................................................................................... 3

LIST OF ABBREVIATIONS.............................................................................................................. 5

CHAPTER I REVIEW OF THE LITERATURE............................................................................. 7

I.1 Short introduction to the class of Mollicutes with emphasis on the genus -

Mycoplasma .................................................................................................................... 8

I.1.1 Phylogeny and taxonomy.......................................................................................... 9

I.1.2 Genome structure and organisation ........................................................................ 12

I.1.2.1 Genome sizes and sequence projects ............................................................ 12

I.1.2.2 General concepts........................................................................................... 12

I.1.3 Role of mollicutes in diseases................................................................................. 15

I.1.3.1 Repetitive elements in mycoplasmas: antigenic size- and phase-

variation ........................................................................................................ 15

I.1.3.2 Diseases related to mycoplasmas.................................................................. 16

I.2 Molecular techniques to detect, identify & type mycoplasmas.................................... 22

I.2.1 Introduction............................................................................................................. 23

I.2.1.1 Definitions .................................................................................................... 23

I.2.1.2 Molecular techniques for the detection and identification of

mycoplasmas................................................................................................. 23

I.2.1.3 Molecular techniques for the typing of Mycoplasma strains........................ 24

I.2.1.4 Classification of the described molecular techniques................................... 25

I.2.2 Molecular techniques performed on the entire Mycoplasma genome .................... 26

I.2.2.1 Based on restriction ...................................................................................... 26

I.2.2.2 Based on restriction with hybridisation ........................................................ 29

I.2.2.3 Based on restriction and PCR ....................................................................... 32

I.2.2.4 Based on PCR ............................................................................................... 33

I.2.3 Molecular techniques performed on defined chromosomal loci............................. 35

I.2.3.1 Based on hybridisation.................................................................................. 35

I.2.3.2 Based on PCR ............................................................................................... 35

I.2.4 Future techniques & conclusion.............................................................................. 45

CHAPTER II AIMS........................................................................................................................... 65

CHAPTER III EXPERIMENTAL STUDIES ................................................................................. 69

III.1 Evaluation of amplified rDNA restriction analysis (ARDRA) for the

identification of Mycoplasma species......................................................................... 70

III.2 Evaluation of tDNA-PCR for the identification of Mollicutes ................................. 101

III.3 A multiplex PCR to identify porcine mycoplasmas present in broth cultures.......... 123

III.4 Diversity of Mycoplasma hyopneumoniae within and between herds using

Pulsed-Field Gel Electrophoresis.............................................................................. 135

III.5 Comparison of molecular techniques for the typing of

Mycoplasma hyopneumoniae isolates....................................................................... 149

CHAPTER IV GENERAL DISCUSSION..................................................................................... 171

SUMMARY....................................................................................................................................... 185

SAMENVATTING........................................................................................................................... 189

DANKWOORD ................................................................................................................................ 193

CURRICULUM VITAE .................................................................................................................. 197

LIST OF ABBREVIATIONS

(k)bp (kilo) base pair

AFLP Amplified fragment length polymorphism analysis

AP-PCR Arbitrarily primed PCR

ARDRA Amplified rDNA restriction analysis

ATCC American Type Culture Collection

BLAST Basic local alignment search tool

CCU Colour changing units

CFU Colony forming units

CHEF Contour-clamped homogeneous electric field

CIRAD Agricultural research centre for international development (Montpellier, France)

CODA Centrum voor onderzoek in de diergeneeskunde en agrochemie (Ukkel, Belgium)

dbp differential base pair

DFVF Danish institute for food and veterinary research (Copenhagen, Denmark)

DGGE Denaturing gradient gel electrophoresis

DNA Desoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

FISH Fluorescent in situ hybridisation

IS Insertion sequence

GUH Ghent university hospital

ITG Institute of Tropical Diseases (Antwerp, Belgium)

ITS Intergenic (transcribed) spacer(s)

MW Molecular weight

NCTC National Collection of Type Cultures

NHS20 Friis’ broth

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PFGE Pulsed field gel electrophoresis

PRDC Porcine respiratory disease complex

RAPD Randomly amplified polymorphic DNA

rDNA Ribosomal DNA

REA Restriction endonuclease analysis

RFLP Restriction fragment length polymorphism

RNA Ribonucleic acid

RT Reverse transcriptase

sp. species (singular)

spp. species (plural)

ssp. (subsp.) subspecies (singular)

sspp. subspecies (plural)

tRNA transfer RNA

UPGMA Unweighted pair group method with arithmetic means

VUB Free university of Brussels

VNTR Variable number of tandem-repeats

7

CHAPTER I

Review of the

Literature

8 Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma

I.1 SHORT INTRODUCTION TO THE CLASS OF MOLLICUTES

WITH EMPHASIS ON THE GENUS MYCOPLASMA

Mollicutes evolved from AT-rich, gram-positive bacteria to become the smallest self-

replicating organisms known to date. During their degenerative evolution, their genomes

considerably reduced in size and many genes, common to most bacteria, were lost. Most

characteristically, mollicutes (mollis = soft, cutis = skin) lost the genes involved in the

synthesis of a cell wall. The presence of a cell membrane as only boundary implies an

intrinsic resistance to antimicrobial agents that inhibit cell wall synthesis, a sensitivitity to

osmotic shock and an ability to pass filters typically used to sterilise solutions. Moreover,

because of their small genomes, mollicutes have limited biosynthetic capabilities and occur as

obligate parasites in a wide diversity of plant and animal hosts. Thus far, audacious efforts

have led to the description of already about 200 species, and still, this number likely

represents only a minor fraction of the mollicutes present in nature. Of the eight genera

currently described within the class of Mollicutes, the genus Mycoplasma is by far most

studied. We will therefore especially focus on this latter group of bacteria, describe their

taxonomic position and enlighten some interesting features related to their small genomes.

Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma 9

I.1.1 Phylogeny and taxonomy Initial data about mollicutes were rather confusing since terms as viruses, L-forms, or

pleuropneumonia-like agents were used to describe these organisms. The first mollicute, later

acknowledged as Mycoplasma mycoides subsp. mycoides small colony (SC), was isolated and

described in 1898, but it took another few decades before other animal mycoplasmas were

found (5). For instance, the porcine pathogen M. hyopneumoniae was only demonstrated in

1965 (28). The first human mycoplasma, M. pneumoniae, was described in 1937 (14). Related

mycoplasma-like organisms infecting plants and insects were only discovered in 1967 (29).

Currently, these bacteria comprise the class of Mollicutes (Table 1). Typically, they are

divided in eight different genera. An additionally genus of phytoplasmas is not officially

acknowledged since all in vitro cultivation steps were so far unsuccessful.

Phylogenetic data based on 16S rRNA gene sequences confirm the taxonomic status of the

Mollicutes closely related to gram-positve bacteria as they likely split up from the

Streptococcus phylogenetic branch about 600 million years ago (27). However, within the

class itself, the taxonomic position of several species does not accord with phylogenetic data

and already a number of reclassifications for the class of Mollicutes have been proposed (6,

7). Especially the taxonomy of the genus Mycoplasma is confusing (Figure 1). At the

moment, the genus Mycoplasma encompasses a pneumoniae group, a spiroplasma or

mycoides group, a hominis group, and an anaeroplasma group (comprising only one species).

The Haemobartonella and Eperythrozoon species were recently correctly placed within the

genus Mycoplasma (32), but also currently enlisted Mycoplasma species should be

reclassified to other genera. The current problems are probably best exemplified by the

taxonomic position of the M. mycoides cluster. Unequivocal evidence places this cluster more

closely to spiroplasmas than to mycoplasmas (18). However, owing to practical and

legislative complications, taxonomic changes have not been realised (6) and one species of

the M. mycoides cluster, referred to as Mycoplasma bovine group 7 and closely related to M.

capricolum sspp., has still no official name.


Table 1: Taxonomy and major features of members of the class of Mollicutes.

Classification Order Family Genus

Genome size (kbp)

%GC Cholesterol requirement

O21 Habitat

Mycoplasmatales Mycoplasmataceae Mycoplasma 580-1350 23-40 Yes FA animals and

humans Ureaplasma 760-1170 27-30 Yes FA animals and

humans Entomoplasmatales Entomoplasmataceae Entomoplasma 790-1140 27-29 Yes FA plants and insects Mesoplasma 870-1100 27-30 No FA plants and insects Spiroplasmataceae Spiroplasma2 940-2220 25-31 Yes FA plants and insects Acholeplasmatales Acholeplasmataceae Acholeplasma 1500-1650 25-36 No FA animals, plants and

insects Phytoplasma3 640-1185 23-29 ND ND plants and insects Anaeroplasmatales Anaeroplasmataceae Anaeroplasma 1500-1600 29-34 Yes OAN bovine and ovine

rumen Asteroleplasma 1500 40 No OAN bovine and ovine

rumen 1 O2 = oxygen requirement; FA = facultative aerobe; OAN = obligate anaerobe; ND = not determined. 2 Spiroplasmas have a coiled (spiral) morphology 3 Phytoplasmas have not been cultured and, therefore, have no official taxonomic status, although they are close related

to the Acholeplasma. These microorganisms are likely obligate intracellular parasites.


Figure 1: A phylogenetic tree of

Mycoplasma spp. based on their 16S rDNA

sequences. The tree was computed using

the neighbour-joining algorithm (Mega 2.1

software package). Distances were

corrected for multiple substitutions at

single locations by the one-parameter

model of Jukes and Cantor (transition /

transversion ratio set to 2). Bootstrap

percentages obtained from 500 resampling

steps are indicated at the nodes. The major

groups are indicated by a Roman number,

followed by an Arabic letter for the clusters

named by their representative species

according an earlier rapport of Johansson

and Pettersson (25).

I anaeraoplasma group II hominis group

a: bovis b: lipophilum c: synoviae d: equigenitalium e: pulmonis f: gypsies g: hominis h: sualvi i: neurolyticum

III pneumoniae group

a: haemotrophic mollicutes b: fastidiosum c: muris d: pneumoniae

IV spiroplasma group


I.1.2 Genome structure and organisation

I.1.2.1 Genome sizes and sequence projects

A drastic economisation in genetic information is common to mollicutes and sizes ranging

from 580 kbp (M. genitalium) to 2220 kbp (Spiroplasma ixodetis) have been reported (10,

36). For several of these mollicutes, the genome sequence has already been fully or partially

determined (Table 2). The M. genitalium genome even appeared only a few months after that

of Haemophilus influenzae, the first fully sequenced bacterial genome. Its genome comprises

only around 500 predicted genes (compared to about 4000 in E. coli) and since it is still the

smallest bacterial genome discovered, M. genitalium is often used as a reference organism to

describe the ‘minimal gene concept enabling life’ (17, 23). Besides several fully sequenced

mycoplasmas, also the genome sequence of Ureaplasma parvum, Candidatus Phytoplasma

asteris and Mesoplasma florum were determined (Table 2). The genome sequences of

Spiroplasma kunkulii, Spiroplasma citri and many other mollicutes will follow soon. This

wealth of information will lead to a further understanding of the evolution, biochemical

pathways and characteristics of mollicutes in general. However, since mycoplasmas are still

by far more studied, the here presented genomic data will mainly focus on this genus and will

only in some occasions be complemented with data of other mollicute genera.

I.1.2.2 General concepts

Apart from having no cell wall, the genome reduction of mycoplasmas (and mollicutes in

general) is associated with moderate anabolic capabilities. They have no genes involved in

amino acid biosynthesis and only a few genes involved in the biosynthesis of cofactors

(vitamins) (35). Most mycoplasmas cannot synthesise any fatty acids and some even

incorporate exogeneous phospholipids together with cholesterol in their cell membrane. Also

the genes involved in the biosynthesis of nucleotides are very limited (35). Their parasitic

lifestyle coincides with a significant number of mycoplasmal genes devoted to proteases, but

only a small number of genes encoding transport systems. These findings may possibly be

explained by the low substrate specificity of the systems and/or the fact that only one barrier

must be crossed.

Besides limited de novo synthesis pathways, their genomes carry a minimal set of genes

involved in energy metabolism. The use of carbohydrates is inefficient since both the

tricarboxylic acid cycle and cytochromes are missing. Substrate-phosphorylation is the major

route for ATP synthesis. Mycoplasmas mostly depend on the glycolysis for ATP, although


sometimes other energy providing pathways, like the dihydrolysation of arginine, are assumed

important. In ureaplasmas the hydrolysis of urea is assumed the major pathway for ATP

synthesis (19). Probably mollicutes need less energy for their limited anabolic activity. This

hypothesis is further substantiated by genomic data of Candidatus Phytoplasma asteris. Since

phytoplasmas live intracellularly with an easy access to nutrients, they have seemingly even

fewer genes related to metabolic functions and ATP-synthesis processes (33).

The degenerative evolution of mycoplasmas can also be observed from their number of genes

related to DNA replication, transcription and translation. Mycoplasmas use a simplified DNA

replication complex resembling polymerase III of gram-positive bacteria. Another polymerase

without proofreading activity, resembling PolC of E. coli, has been described as well (1). The

number of genes with respect to DNA repair systems is much lower compared to other

bacteria and the missing or ineffective uracil-DNA glycosylase may explain the low GC-

content (typically lower than 35%) (19, 49). The DNA-dependent RNA polymerase in

mycoplasmas is similar to those found in other bacteria. However, the regulation of

differences in gene expression is largely unknown since, in contrary to most other bacteria,

only one single sigma factor was found (4). Also translation is carried out using a minimal set

of genes. Mycoplasmas contain no more than 1 or 2 copies of rrn operons and only around 30

tRNA genes (12). Interestingly, mycoplasmas (and most, but not all mollicutes) have a tRNA

gene that translates the UGA codon into tryptophan, instead of recognizing it as a stop codon.

Possibly owing to their low GC-content, this UGA codon is far more frequently used than

their cognate UGG codon (48). Overall, the reduced number of genes involved in DNA

replication, transcription and translation coincide with a reduced replication rate. It may be

noteworthy however, that although the number of these genes is low, the relative percentage

in the genome is higher compared to other bacteria, indicating their importance.


Table 2: Completed and ongoing genome sequence projects of Mollicutes species1.

Mollicutes species (website)

Genome size (bp)

Strain Genbank Accesssion Number

Ref.

Candidatus Phytoplasma asteris 860631 Onion Yellows NC_005303 (33) (http://papilio.ab.a.u-tokyo.ac.jp/planpath/phyto-genome/index.html)

Mesoplasma florum L1 NC_006055 (http://www.broad.mit.edu/annotation/microbes/mesoplasma_florum)

Mycoplasma alligatoris A21JP2 (http://www.biotech.ufl.edu/Genomics/index.html)

Mycoplasma arthritidis 820453 158L3-1 NC_004819 (http://www.tigr.org/tdb/mdb/mdbinprogress.html#codes)

Mycoplasma bovis PG45 (http://www.tigr.org/tdb/mdb/mdbinprogress.html#codes)

Mycoplasma capricolum subsp. capricolum California Kid (http://www.tigr.org/tdb/mdb/mdbinprogress.html)

Mycoplasma fermentans M64 (http://gel.ym.edu.tw/projects/mycoplasma/index.html)

Mycoplasma gallisepticum 996422 R NC_004829 (34) (http://cevr.uconn.edu/genome.html)

Mycoplasma genitalium 580070 G37 NC_000908 (17) (http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=gmg)

Mycoplasma hyopneumoniae 892758 USA232 NC_006360 (31) (http://mycoplasmas.vm.iastate.edu/seq/Mhyo.html)

Mycoplasma mobile 777079 163K NC_006908 (24) (http://www.broad.mit.edu/annotation/microbes/mycoplasma/)

Mycoplasma mycoides subsp. mycoides SC 1211703 PG1T NC_005364 (48) (http://www.biotech.kth.se/molbio/key_achievements/mycoplasma.html)

Mycoplasma penetrans 1358633 HF-2 NC_004432 (41) (http://www.nih.go.jp/Mypet)

Mycoplasma pneumoniae 816394 M129 NC_000912 (21) (http://www.zmbh.uni-heidelberg.de/M_pneumoniae/genome/Results.html)

Mycoplasma pulmonis 963879 UAB CTIP NC_002771 (11) (http://genolist.pasteur.fr/MypuList/help/project.html)

Mycoplasma synoviae (http://www.brgene.lncc.br/indexMS.html)

Sprioplasma kunkelii 1495357 CR2-3x NC_003999 (http://www.genome.ou.edu/spiro.html)

Ureaplasma parvum 751719 ATCC 70970 NC_002162 (19) (http://genome.microbio.uab.edu/uu/uugen.htm)

1 The table is last updated in March 2005 and blank fields represent yet unavailable data


I.1.3 Role of mollicutes in diseases Most mollicutes are not linked to any disease so far and even beneficial effects on the survival

of insect hosts have been described for some plant pathogenic mollicutes (3). The

pathogenicity of other mollicutes is unequivocal and their virulence mechanisms are

undeniably complex. For the male-killing Spiroplasma poulsonii found in Drosophila

melanogaster, the molecular details involving a dosage compensation complex were recently

described (47), but for most other mollicutes, the determination of virulence factors is slowly

progressing. Even for well-studied mycoplasmas, the exact way in which they cause disease is

still confusing as they likely challenge the host’s immune response differently compared to

more common bacterial pathogens. The ability of mycoplasmas to induce a broad range of

immunoregulatory events certainly contributes to the existing controversy. Cytokine

production and immune cell activation may trigger immunosuppression, or as often seen, may

provoke the host immune response and result in lesion development (or auto-immunity) (26,

40). Adherence to the host cell plays an important role and is a prerequisit not only for the

mycoplasma parasitic mode of life, but also for their virulence (39). The discovery of various

genetic systems enabling attachment to host cells and providing a highly versatile surface coat

is suggested an important mechanism to escape the host immune response (13). Some

mycoplasmas have even been reported to invade non-phagocytic cells (41, 50, 51). It should

be emphasised that this intracellular location, even for a short period, may render

mycoplasmas less vulnerable to the host immune response or may result in long term survival

during antibiotic treatment (37).

I.1.3.1 Repetitive elements in mycoplasmas: antigenic size- and phase-variation

Mycoplasma genomes contain a remarkably high percentage of repetitive sequences (38). For

instance, repetitive elements composed of short segments of the MgPa adhesin of M.

genitalium are distributed over the genome and constitute, together with the intact MgPa

operon, 4.7% of the total genome size (17). An even larger number of vlhA (or pMGA) genes

in M. gallisepticum occupy no less than 10.4% of the genome (34). The total number of

repeats in M. mycoides subsp. mycoides SC constitutes even 29%, the highest percentage of

repetitive sequences in any bacterial genome so far (48). The presence of these elements

seems to contradict with the expectation of a minimal genome indicating a strong selective

pressure for their remanence (30). This may be clarified by the role they play in the antigenic


variation of the cell surface (46). The discovery of this antigenic size- and phase-variation is

likely one of the major advances in mycoplasma research.

Several different mechanisms have been described for the size- and/or phase-variation of

surface lipoproteins. In several mycoplasmas, multiple copies of a particular lipoprotein

encoding gene are present, but neither the gene order, nor the number of repeated units are

conserved between different strains. At one specific moment in time, one particular gene copy

succeeding the only active promotor will be expressed, while all other copies remain silent.

Recombination processes allow alterations between the expressed genes. Studies on the vsa

locus of M. pulmonis led to the identification of a site-specific DNA recombinase responsible

for such phase-variable production of surface lipoproteins (44) and probably similar

mechanisms exist for the vsp locus of M. bovis and the vmpa locus of M. agalactiae (16, 20).

Antigenic varation due to the presence of inverted promoters or polymerase slippage has been

described as well (22, 52). Another system, involving post-translational modification, was

shown in M. hyopneumoniae and M. fermentans to be strain-specific and may additionally be

used to deceive the host immune response (9, 15). Apart from evasion, the number of repeats

may also have direct impact on the host immune response. In case of M. pulmonis, the

number of tandem repeats in the VsaA protein was linked to resistance to complement

activation (42, 43), while in M. arthritidis, a difference of the number of tandem repeats was

directly linked to virulence (45). This indicates that phase- or size variation may have key-

roles in virulence, host defence and adherence of mycoplasmas in general.

I.1.3.2 Diseases related to mycoplasmas

Although mycoplasma infections are typically of a slumbering, chronic nature, some

infections may evolve rapidly and are both deadly and highly contagious. Notably, the first

isolated mycoplasma, namely M. mycoides subsp. mycoides SC, is from a global perspective

still considered one of the most important bovine diseases. M. alligatoris and M. crocodyli are

highly virulent as well and can kill alligators, caimans or crocodiles within a week, which is

remarkably fast for mycoplasma related diseases (8). M. gallisepticum causes important losses

in chicken flocks and is even more virulent to turkeys. Although other examples exist,

mycoplasma infections are rarely deadly.

Not seldom, mycoplasmas act as primary pathogens or as a cofactor in more severe diseases.

For instance, M. hyopneumoniae is the primary agent of enzootic pneumonia, which is one of

the most important respiratory diseases in pigs. Since infected pigs are more vulnerable,


secondary infections often aggrevate the disease and lead to more clinical symptoms. Also

more and more provoking reports link mycoplasmas as cofactor in complex diseases like

AIDS, Crohn’s disease or rheumatoid arthritidis (2), but the intruiging role of mycoplasmas in

these complex diseases leaves room for debate.

References

1. Barnes, M. H., P. M. Tarantino, Jr., P. Spacciapoli, N. C. Brown, H. Yu, and K. Dybvig. 1994.

DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its

structural gene, polC. Mol. Microbiol. 13:843-854.

2. Baseman, J. B., and J. G. Tully. 1997. Mycoplasmas: sophisticated, reemerging, and burdened by

their notoriety. Emerg Infect Dis. 3:21-32.

3. Beanland, L., C. W. Hoy, S. A. Miller, and L. R. Nault. 2000. Influence of aster yellows

phytoplasma on the fitness of aster leafhopper (Homoptera: Cicadellidae). Ann. Entomol. Soc. Am.

93:271-276.

4. Bornberg-Bauer, E., and J. Weiner, 3rd. 2002. A putative transcription factor inducing mobility in

Mycoplasma pneumoniae. Microbiology. 148:3764-3765.

5. Bové, J. M. 1999. The one-hundredth anniversary of the first culture of a mollicute, the contagious

bovine peripneumonia microbe, by Nocard and Roux, with the collaboration of Borrel, Salimbeni, and

Dujardin-Baumetz. Res. Microbiol. 150:239-245.

6. Bradbury, J. M. 1997. International committee on systematic bacteriology, subcommittee on the

taxonomy of Mollicutes. Int. J. Syst. Evol. Microbiol. 47:911-914.



8. Brown, D. R., L. A. Zacher, and W. G. Farmerie. 2004. Spreading factors of Mycoplasma

alligatoris, a flesh-eating mycoplasma. J. Bacteriol. 186:3922-3927.

9. Calcutt, M. J., M. F. Kim, A. B. Karpas, P. F. Muhlradt, and K. S. Wise. 1999. Differential

posttranslational processing confers intraspecies variation of a major surface lipoprotein and a

macrophage-activating lipopeptide of Mycoplasma fermentans. Infect. Immun. 67:760-771.

10. Carle, P., F. Laigret, J. G. Tully, and J. M. Bove. 1995. Heterogeneity of genome sizes within the

genus Spiroplasma. Int. J. Syst. Evol. Microbiol. 45:178-181.

11. Chambaud, I., R. Heilig, S. Ferris, V. Barbe, D. Samson, F. Galisson, I. Moszer, K. Dybvig, H.

Wroblewski, A. Viari, E. P. Rocha, and A. Blanchard. 2001. The complete genome sequence of the

murine respiratory pathogen Mycoplasma pulmonis. Nucleic Acids Res. 29:2145-2153.


12. Dandekar, T., M. Huynen, J. T. Regula, B. Ueberle, C. U. Zimmermann, M. A. Andrade, T.

Doerks, L. Sanchez-Pulido, B. Snel, M. Suyama, Y. P. Yuan, R. Herrmann, and P. Bork. 2000. Re-

annotating the Mycoplasma pneumoniae genome sequence: adding value, function and reading frames.

Nucleic Acids Res. 28:3278-3288.

13. Denison, A. M., B. Clapper, and K. Dybvig. 2005. Avoidance of the host immune system through

phase variation in Mycoplasma pulmonis. Infect. Immun. 73:2033-2039.

14. Dienes, L., and G. Edsall. 1937. Observations on the L-organism of Klieneberger. Proc Soc Exp Biol

Med. 36:740-744.

15. Djordjevic, S. P., S. J. Cordwell, M. A. Djordjevic, J. Wilton, and F. C. Minion. 2004. Proteolytic

processing of the Mycoplasma hyopneumoniae cilium adhesin. Infect Immun. 72:2791-2802.

16. Flitman-Tene, R., S. Levisohn, I. Lysnyansky, E. Rapoport, and D. Yogev. 2000. A chromosomal

region of Mycoplasma agalactiae containing vsp-related genes undergoes in vivo rearrangement in

naturally infected animals. FEMS Microbiol. Lett. 191:205-212.

17. Fraser, C. M., J. D. Gocayne, O. White, M. D. Adams, R. A. Clayton, R. D. Fleischmann, C. J.

Bult, A. R. Kerlavage, G. Sutton, J. M. Kelley, and et al. 1995. The minimal gene complement of

Mycoplasma genitalium. Science. 270:397-403.

18. Gasparich, G. E., R. F. Whitcomb, D. Dodge, F. E. French, J. Glass, and D. L. Williamson. 2004.

The genus Spiroplasma and its non-helical descendants: phylogenetic classification, correlation with

phenotype and roots of the Mycoplasma mycoides clade. Int. J. Syst. Evol. Microbiol. 54:893-918.

19. Glass, J. I., E. J. Lefkowitz, J. S. Glass, C. R. Heiner, E. Y. Chen, and G. H. Cassell. 2000. The

complete sequence of the mucosal pathogen Ureaplasma urealyticum. Nature. 407:757-762.

20. Glew, M. D., M. Marenda, R. Rosengarten, and C. Citti. 2002. Surface diversity in Mycoplasma

agalactiae is driven by site-specific DNA inversions within the vpma multigene locus. J. Bacteriol.

184:5987-5998.

21. Himmelreich, R., H. Hilbert, H. Plagens, E. Pirkl, B. C. Li, and R. Herrmann. 1996. Complete

sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res.

24:4420-4449.

22. Horino, A., Y. Sasaki, T. Sasaki, and T. Kenri. 2003. Multiple promoter inversions generate surface

antigenic variation in Mycoplasma penetrans. J. Bacteriol. 185:231-242.

23. Hutchison, C. A., S. N. Peterson, S. R. Gill, R. T. Cline, O. White, C. M. Fraser, H. O. Smith, and

J. C. Venter. 1999. Global transposon mutagenesis and a minimal mycoplasma genome. Science.

286:2165-2169.


24. Jaffe, J. D., N. Stange-Thomann, C. Smith, D. DeCaprio, S. Fisher, J. Butler, S. Calvo, T. Elkins,

M. G. FitzGerald, N. Hafez, C. D. Kodira, J. Major, S. Wang, J. Wilkinson, R. Nicol, C.

Nusbaum, B. Birren, H. C. Berg, and G. M. Church. 2004. The complete genome and proteome of

Mycoplasma mobile. Genome Res. 14:1447-1461.

25. Johansson, K. E., and B. Pettersson. 2002. Taxonomy of Mollicutes, p. 1-29. In S. Razin, and R.

Herrmann (ed.), Molecular biology an pathogenicity of mycoplasmas. Kluwer Academic/Plenum

Publishers, New York.

26. Jones, H. P., L. Tabor, X. Sun, M. D. Woolard, and J. W. Simecka. 2002. Depletion of CD8+ T

cells exacerbates CD4+ Th cell-associated inflammatory lesions during murine mycoplasma respiratory

disease. J. Immunol. 168:3493-3501.

27. Maniloff, J. 1996. The minimal cell genome: On being the right size. Proc. Natl. Acad. Sci. USA.

93:10004-10006.

28. Mare, C. J., and W. P. Switzer. 1965. New Species: Mycoplasma hyopneumoniae; a causative agent

of virus pig pneumonia. Vet. Med. Small. Anim. Clin. 60:841-846.

29. McCoy, R. E., A. Caudwell, C. J. Chang, T. A. Chen, L. N. Chiykowski, M. T. Cousin, J. L. Dale,

G. T. N. deLeeuw, D. A. Golino, K. J. Hackett, B. C. Kirkpatrick, R. Marwitz, H. Petzold, R. C.

Sinha, M. Sugiura, R. F. Whitcomb, I. L. Yang, B. M. Zhu, and E. Seemüller. 1989. Plant diseases

associated with mycoplasma-like organisms, p. 545-640. In R. F. Whitcomb, and J. G. Tully (ed.),

Spiroplasmas, acholeplasmas, and mycoplasmas of plants and arthropods, vol. vol. 5. Academic Press,

Inc., New York, N.Y.

30. Metzgar, D., L. Liu, C. Hansen, K. Dybvig, and C. Wills. 2002. Domain-level differences in

microsatellite distribution and content result from different relative rates of insertion and deletion

mutations. Genome Res. 12:408-413.

31. Minion, F. C., E. J. Lefkowitz, M. L. Madsen, B. J. Cleary, S. M. Swartzell, and G. G. Mahairas.

2004. The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine

mycoplasmosis. J. Bacteriol. 186:7123-7133.

32. Neimark, H., K. E. Johansson, Y. Rikihisa, and J. G. Tully. 2001. Proposal to transfer some

members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with

descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris',

'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'. Int. J. Syst. Evol.

Microbiol. 51:891-899.

33. Oshima, K., S. Kakizawa, H. Nishigawa, H. Y. Jung, W. Wei, S. Suzuki, R. Arashida, D. Nakata,

S. Miyata, M. Ugaki, and S. Namba. 2004. Reductive evolution suggested from the complete genome

sequence of a plant-pathogenic phytoplasma. Nat. Genet. 36:27-29.


34. Papazisi, L., T. S. Gorton, G. Kutish, P. F. Markham, G. F. Browning, D. K. Nguyen, S. Swartzell,

A. Madan, G. Mahairas, and S. J. Geary. 2003. The complete genome sequence of the avian

pathogen Mycoplasma gallisepticum strain R(low). Microbiology. 149:2307-2316.

35. Pollack, J. D. 2002. The necessity of combining genomic and enzymatic data to infer metabolic

function and pathways in the smallest bacteria: amino acid, purine and pyrimidine metabolism in

Mollicutes. Front. Biosci. 7:d1762-1781.

36. Pyle, L. E., L. N. Corcoran, B. G. Cocks, A. D. Bergemann, J. C. Whitley, and L. R. Finch. 1988.

Pulsed-field electrophoresis indicates larger-than-expected sizes for mycoplasma genomes. Nucleic

Acids Res. 16:6015-6025.

37. Reinhardt, A. K., A. V. Gautier-Bouchardon, M. Gicquel-Bruneau, M. Kobisch, and I. Kempf.

2005. Persistence of Mycoplasma gallisepticum in chickens after treatment with enrofloxacin without

development of resistance. Vet. Microbiol. 106:129-137.

38. Rocha, E. P., and A. Blanchard. 2002. Genomic repeats, genome plasticity and the dynamics of

Mycoplasma evolution. Nucleic Acids Res. 30:2031-2042.

39. Rosengarten, R., C. Citti, M. Glew, A. Lischewski, M. Droesse, P. Much, F. Winner, M. Brank,

and J. Spergser. 2000. Host-pathogen interactions in mycoplasma pathogenesis: virulence and survival

strategies of minimalist prokaryotes. Int. J. Med. Microbiol. 290:15-25.

40. Rottem, S. 2003. Interaction of mycoplasmas with host cells. Physiol Rev. 83:417-32.

41. Sasaki, Y., J. Ishikawa, A. Yamashita, K. Oshima, T. Kenri, K. Furuya, C. Yoshino, A. Horino, T.

Shiba, T. Sasaki, and M. Hattori. 2002. The complete genomic sequence of Mycoplasma penetrans,

an intracellular bacterial pathogen in humans. Nucleic Acids Res. 30:5293-5300.

42. Simmons, W. L., A. M. Denison, and K. Dybvig. 2004. Resistance of Mycoplasma pulmonis to

complement lysis is dependent on the number of Vsa tandem repeats: shield hypothesis. Infect. Immun.

72:6846-6851.

43. Simmons, W. L., and K. Dybvig. 2003. The Vsa proteins modulate susceptibility of Mycoplasma

pulmonis to complement killing, hemadsorption, and adherence to polystyrene. Infect. Immun.

71:5733-5738.

44. Sitaraman, R., A. M. Denison, and K. Dybvig. 2002. A unique, bifunctional site-specific DNA

recombinase from Mycoplasma pulmonis. Mol. Microbiol. 46:1033-1040.

45. Tu, A. H., B. Clapper, T. R. Schoeb, A. Elgavish, J. Zhang, L. Liu, H. Yu, and K. Dybvig. 2005.

Association of a major protein antigen of Mycoplasma arthritidis with virulence. Infect. Immun.

73:245-249.

46. van der Woude, M. W., and A. J. Baumler. 2004. Phase and antigenic variation in bacteria. Clin.

Microbiol. Rev. 17:581-611.


47. Veneti, Z., J. K. Bentley, T. Koana, H. R. Braig, and G. D. Hurst. 2005. A functional dosage

compensation complex required for male killing in Drosophila. Science. 307:1461-1463.

48. Westberg, J., A. Persson, A. Holmberg, A. Goesmann, J. Lundeberg, K. E. Johansson, B.

Pettersson, and M. Uhlen. 2004. The genome sequence of Mycoplasma mycoides subsp. mycoides SC

type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res.

14:221-227.

49. Williams, M. V., and J. D. Pollack. 1990. A mollicute (mycoplasma) DNA repair enzyme:

purification and characterization of uracil-DNA glycosylase. J. Bacteriol. 172:2979-2985.

50. Winner, F., R. Rosengarten, and C. Citti. 2000. In vitro cell invasion of Mycoplasma gallisepticum.

Infect. Immun. 68:4238-4244.

51. Yavlovich, A., M. Tarshis, and S. Rottem. 2004. Internalization and intracellular survival of

Mycoplasma pneumoniae by non-phagocytic cells. FEMS Microbiol. Lett. 233:241-246.

52. Zhang, Q., and K. S. Wise. 1997. Localized reversible frameshift mutation in an adhesin gene confers

a phase-variable adherence phenotype in mycoplasma. Mol. Microbiol. 25:859-869.

22 Molecular techniques to detect, identify & type mycoplasmas

I.2 MOLECULAR TECHNIQUES TO DETECT, IDENTIFY & TYPE

MYCOPLASMAS

Mycoplasmas, the smallest self-replicating life forms, are primarily characterised by their lack

of a cell wall and cholesterol containing membrane. Despite their fastidious nature, an

extensive number of mycoplasmas have already been described and more are discovered

annually. Conventional methods for the detection and identification of mycoplasmas

systematically involve enrichment steps in selective broth followed by morphological,

biochemical and serological tests. Although well established, these techniques have some

important drawbacks. The morphological and biochemical characteristics are in general not

discriminative, while serological cross-reactions have been frequently reported as well.

Moreover, these classical techniques are often labour intensive and hardly ever useful to

differentiate strains belonging to the same species. The advent of molecular biology has

greatly enhanced the capability to detect and identify species, to classify and characterise

strains and to assess the genetic diversity of populations. The aim of this review is to provide

an overview of these molecular techniques and their applications in the field of

mycoplasmology.

Molecular techniques to detect, identify & type mycoplasmas 23

I.2.1 Introduction

I.2.1.1 Definitions

In the light of evolution, it may not always be clear at what point a population of cells belong

to one particular or to more related ecotypes or species. Bacteria are ever evolving and the

term species leaves room for debate and is subject to change (164). Consequently, the terms

identification and typing are frequently used interchangeably. Nevertheless, if possible, we

will use identification solely for differences at species-level or to discriminate acknowledged

subspecies, and typing for the differentiation of strains. Hereby, a strain or isolate is defined

as cultures or subcultures derived from a single pure colony. The term clone is used in a rather

wider context and denotes isolates that are indistinguishable in genotype at which the most

likely explanation is a common ancestor (34). The definition of detection, as revealing what

was concealed or hidden, is in our context mainly used to demonstrate the presence of

mycoplasmas. Although straightforward, the term identification in literature often

automatically includes detection as well, especially if achieved simultaneously using only one

technique.

I.2.1.2 Molecular techniques for the detection and identification of mycoplasmas

The small genomes of mycoplasmas lack many essential genes. As a consequence,

mycoplasmas are found as obligate parasites in a wide variety of animal hosts, including

humans. Depending on the species, the impact on the host may vary. Some mycoplasmas may

reside (seemingly) unnoticed, while others are highly virulent. Most typically, they cause

infections of a more chronic nature and may act as primary agents, enabling opportunistic

bacteria to infect the more vulnerable host. This may complicate diagnosis and the role of

mycoplasmas is often overlooked. Furthermore, a correct detection and identification by

traditional tests is complicated by the slow growth of mycoplasmas, the necessity of complex

isolation media and the limited discriminatory power for the continuously growing number of

Mycoplasma species and subspecies.

The development of more accurate and faster techniques has become increasingly important

and although some generally applicable tests have been described (e.g. 129), especially

molecular biology opened a path to shorten detection times and to improve identification

methods. In particular PCR methods appear very promising to replace more and more

conventional methods, although further improvements are necessary (197). The current PCRs


often work perfectly on purified samples, but their usefulness for detection and simultaneous

identification may decrease dramatically when applied to biological materials. The

importance of sample pre-processing or DNA extraction methods, which may need case-to-

case optimisation to yield compatible results (150), are hard to standardise and selective

enrichment steps are frequently preferred. Moreover, PCR tests are mostly very specific and,

as a consequence, only valuable to detect and/or identify one or a few species. These

problems clarify why quality-controlled, low-cost, commercially available, generally

applicable PCR kits are still rarely available for the detection and identification of

mycoplasmas. With the exception of some commercial kits for mycoplasmas in cell-cultures

(156), institutes must rely on in-house protocols. However, as demonstrated for M.

pneumoniae (109), the compliance of these protocols is often low and the necessary validation

studies to prove their usefulness are often missing. Nonetheless, molecular techniques are

likely to be an increasingly important tool for the detection and identification of bacteria in

the future, especially for fastidious bacteria like Mollicutes.

I.2.1.3 Molecular techniques for the typing of Mycoplasma strains

In contrary to the popularity of DNA based identification methods, typing methods are only

gradually implemented in the field of mycoplasmology. Ideally, every typing method should

be validated using multiple strains from different geographical regions or epidemiological

episodes. Unfortunately, these are rarely available for mycoplasmas. Firstly, for some species,

the isolation is often merely too complex and laborious to perform. Secondly, some mildly or

non-pathogenic species are ubiquitously present and not linked to true outbreaks, making

epidemiological episodes hard, if not impossible, to define (77). Finally, some well

documented, important species, like M. pneumoniae, are very homogeneous and difficult or

impossible to type with techniques commonly used for other mycoplasmas (36, 174).

Although not all problems are easily solved, a number of approaches may prove beneficial.

Further optimisation and especially standardisation of current protocols will be increasingly

important to set up collaborative studies between laboratories. An increase in techniques that

yield easily interpretable data that can be stored online may be additionally helpful. For some

bacteria, online databases are already available (MLST-net, PulseNet, …) and it would be

interesting to centralise typing data for some widespread, fastidious mycoplasmas as well. It

is also essential to further develop new molecular techniques and to further exploit the

exponential increase in genomic data. Possibly, the extensive number of repeats present in the


genomes of mycoplasmas (158) may offer interesting alternatives to the already developed

techniques.

Since numerous detailed reports about intraspecific variability appear, it will become

increasingly important to type strains or to define specific subgroups within a species. This

increasing interest in epidemiological data of mycoplasmas will help to elucidate the genomic

plasticity, as observed for some species, to reveal the geographical spreading or transmission

patterns, or to interpret differences seen on the biological level. Ultimately, the understanding

of epidemiological behaviour may offer possibilities to control or even to eradicate

mycoplasma related diseases.

I.2.1.4 Classification of the described molecular techniques

The described molecular techniques can be divided in different categories based on technical

aspects (175, 194). Since extrachromosomal elements are extremely rare in Mycoplasma spp.

(51), we have chosen to divide the techniques into two big subcategories depending on

whether the techniques focus on the entire chromosome or only on specific, well-identified

DNA fragments. For the first category, when examining the entire chromosome, we have

chosen to include all techniques based on restriction of chromosomal DNA. Restricted

fragments can be visualised directly after electrophoresis or indirectly after hybridisation with

specific probes. A second genome wide approach includes a single PCR step to generate a

variable number of fragments.

When only looking at specific genomic fragments, a subdivision can be made whether the

fragments are visualised using hybridisation or whether they are amplified by PCR. The PCR

amplification of these specific fragments is often used for detection and identification, but can

be followed by a restriction analysis to generate (sub)species- or strain-specific fingerprints.

Moreover, differences in sizes or nucleotide sequences of the amplified fragments can

additionally be used for identification or typing and will be described as well.


I.2.2 Molecular techniques performed on the entire Mycoplasma genome

I.2.2.1 Based on restriction

I.2.2.1.1 Pulsed-Field Gel Electrophoresis (PFGE)

During PFGE, mycoplasma cells are embedded in agarose plugs for lysis in order to obtain

intact genomic DNA. This genomic DNA is subsequently digested with a rare cutting

restriction enzyme to generate about 10 to 20 restriction fragments. With the increasingly

number of available genome sequences of several important Mycoplasma spp. (14), the

expected outcome for the reference strain can be easily calculated in silico. For other

mycoplasmas, formulas to estimate the number of restriction sites have been published (60),

but the finding of a suitable restriction enzyme is, despite the rather uniform GC-content of

mycoplasmas, often a matter of trial and error. Once the genomic DNA is digested, the large

genomic DNA fragments are seperated by electrophoresis using an electrical field that

periodically changes over time (125). The alternations of the applied field must be optimised

to the dimensions of the DNA fragments since separation largely depends on the way the

molecules reorient through the gel (72). The newest PFGE apparatuses use a mathematical

algorithm to calculate standard settings for the fluctuations of the electrical field over a given

seperation window.

Over time, PFGE has turned out to be a highly discriminatory typing technique for many

bacteria, including mycoplasmas. Like all bacterial typing procedures, a standardised protocol

is essential to obtain reproducible results (191). For some bacterial species, such protocols are

available and worldwide obtained profiles are stored in central online databases (133, 169).

These data can be compared with data obtained from own isolates, but care must be taken

when interpreting results. After all, PFGE analyses are not suited to depict phylogenetic trees

without further epidemiological data (38) since different profiles may result from single base

substitutions and small insertion or deletions. This implies that differences seen in PFGE

patterns are not easily linked to antigenic variations or biological functions (94, 180). On the

other hand, the technique is perfectly suited to follow epidemic outbreaks or to track specific

strains and unravel infection patterns. The high cost and the labor-intensive protocols remain

important drawbacks of the technique.

For mycoplasmas, described macro-restriction protocols are still far from standardised and

were initially only performed on one or a few reference strains, especially for the estimation


of genome sizes. The potential of PFGE for the typing of mycoplasmas was first

demonstrated by the publication of different physical maps of several M. hominis strains.

These showed a pronounced intraspecific heterogeneity for both PFGE patterns and overall

genome sizes, in spite of considerable gene order conservation (102). A few years later, PFGE

analysis on more M. hominis strains confirmed these results and showed a high diversity

between strains of different origins. When on the other hand strains from individual women

were examined over a 18 month period, nearly identical PFGE patterns were observed (86).

These results are in great similarity to those observed for M. hyosynoviae in pigs where,

despite the extensive intraspecific variety, identical PFGE patterns were observed for at least

two strains isolated from a same herd (95). Highly diverse PFGE patterns were also observed

for a number of avian mycoplasmas such as M. gallisepticum, M. imitans and M. synoviae

(121, 122), but for these species data on the stability of the PFGE patterns during horizontal

or vertical transmission are lacking.

Apart from transmission patterns, PFGE has been proven valuable to demonstrate subgroups

within several Mycoplasma spp. For instance, the very homogeneous M. pneumoniae strains

are typically subdivided into two distinct clusters. PFGE analyses could even demonstrate

small differences between strains of one of these two subgroups (36). Also PFGE patterns of

different strains of M. fermentans clustered, despite extensive intraspecific heterogeneity,

clearly in two distinct groups in accordance with a large difference in genome size (161).

Similarly, for M. bovis isolates, two genetically distinct clusters were apparent using PFGE

(98, 127), possibly representing two clonal lineages with an old ancestral origin. Interestingly,

for the M. bovis isolates, a great intraspecific heterogeneity was observed (98, 127), while

very closely related M. agalactiae strains had exceptionally homogeneous PFGE patterns

(163, 180). M. mycoides subsp. mycoides SC and M. capricolum subsp. capripneumoniae are

two other examples for which PFGE profiles were shown to be very homogeneous, assuming

a strong genomic conservation for these species as well (99).


Table 3: Described PFGE analyses together with the used restriction endonucleases and

number of restriction fragments for the typing or genome size estimation of Mycoplasma

species.

Mycoplasma species Described restriction enzymes (sites) Estimated genome size by PFGE (kbp)

References

M. agalactiae SmaI (7), EclXI (7), BsiWI (3), MluI (8), BssHII (10), SalI (10), XhoI (6), NruI (11), BglI (30), SpeI (30), AvaI (11), MluNI (16), NarI (24), SspI (25), SfuI (23), BlnI (15), AviII (15), SnaBI (30), NaeI (13), SwaI (25), SexAI (25)

945 (NCTC 10123) (180-182)

M. arginini XmaIII (5), SmaI (3) 685 (R16) (7)

M. bovis SmaI (4-8), EclXI (7), BsiWI (3), MluI (7), BssHII (10), SalI (12), XhoI (4), NruI (15), ApaI (3), BglI (30), SpeI (40), AvaI (12), MluNI (18), NarI (20), SspI (30), SfuI (27), BlnI (12), AviII (15), SnaBI (30), NaeI (15), SwaI (25), SexAI (20)

961 (PG45) (98, 181)

M. capricolum subsp. capricolum

BamHI (9), BglI(6), MluI (2), KpnI (3/6), ApaI (2), SalI (2), BssHII (1), SmaI (2), XhoI (2)

1155 (ATCC 27343) (99, 131, 132, 212)

M. capricolum subsp. capripneumoniae

BamHI (7), SmaI (3), HindIII (4) ND1 (99, 131, 132, 212)

M. fermentans SmaI (7), XmaIII (7), BglI, AvaI , BamHI 1035-1130 (K7)

1250-1270 (PG)

(7, 161)

M. flocculare ApaI (10), Asp718 (8), SalI (10) 900 (ATCC 27716) (15)

M. gallisepticum EheI (6), SmaI (5-12), XmaIII (6-7), NarI (6)

1050 (ATCC 19610)

1090 (ATCC 15302)

1130 (5969)

(7, 66, 122, 178)

M. genitalium ApaI (3), MluI, SmaI (8), XhoI (7) 600 (ATCC 33530) (35, 140, 141)

M. haemofelis NruI (28), SalI (21), NotI (9) 1199 (OH) (10, 12)

M. hominis XmaIII (6), ApaLI (7), NarI (4) 675 (strain K)

720 (ATCC 14027)

775 (strain H-34)

(7, 9, 86, 101, 102)

M. hyopneumoniae ApaI (15), ApaLI (13), Asp718 (15), SalI (14)

1070 (ATCC 25934) (16)

M. hyorhinis ApaLI (10) 645 (ATCC 17981) (7)

M. hyosynoviae BssHII (7-13) ND (95)

M. imitans SmaI (5-9) ND (122)

M. iowae - 1280 (68)

M. mobile ApaI (2), MluI (3), BamHI (6), NruI (7) 780 (ATCC 43663) (8)

M. mycoides subsp. mycoides LC

ApaI (2), BamHI (8), BglI (6), BssHII (2), KpnI (3), SalI (3), SmaI (3), XhoI (4)

1200 (Y) (99, 148, 149)

M. mycoides subsp. mycoides SC

BamHI (10), ApaI (2-3), NaeI (2-3), SalI (2-3), SmaI (2-3), XhoI (2-3), Sau3AI (4)

1330 (GC1176-2) (99)

M. orale XmaIII (3), SmaI (3), NarI (6) 680 (ATCC 23714) (7)

M. pneumoniae SfiI (2), ApaI (13) 775-800 (36, 97, 209)

M. salivarium NarI (8) 875 (ATCC 23064) (7)

M. synoviae SmaI (2-12) ND (121) 1 ND = not determined


I.2.2.2 Based on restriction with hybridisation

Before the invention of PFGE, restriction endonuclease analysis (REA) using standard

electrophoresis was proposed as a typing method for mycoplasmas. Although this

straightforward technique seemed promising, the high number of restriction fragments

complicated the interpretation of the data and the technique was rarely used (20, 82, 92). REA

in combination with Southern hybridisation using specific probes enabled the visualisation of

only a limited number of fragments and facilitated the interpretation of these otherwise

complex patterns (218). Initially, probes were radioactively labelled, but nowadays newer and

safer probes based on chemiluminescence (light produced by a chemical reaction) or

fluorescence (energy absorption leading to light) have become available (84). The probe

determines the specificity of the technique and depending on its target, the technique can be

divided in several categories. Hence, our subdivision was made depending on whether probes

target ribosomal, repetitive or other gene sequences. Moreover, the choice of the DNA probe

and restriction endonuclease are crucial for the resolution and the discriminatory index of the

technique. First, restricted genomic DNA fragments ranging from 1-20 kbp in size are usually

preferred (157). Secondly, the presence of restriction sites in the target site will lead to

multiple fragments in the final pattern and will complicate the interpretation (61).

In order to obtain reproducible hybridisation patterns, the starting extracted DNA must be of

high molecular size and free of inhibitors (61). The relatively large quantities necessary are an

extra hindrance when working with fastidious mycoplasmas. Moreover, the procedure of

Southern hybridisation is laborious and, although the technique has been popular for the

identification of mycoplasmas and for the provision of useful insights in the spreading or

transmission of strains, other methods are nowadays preferred.

I.2.2.2.1 With probes based on rDNA sequences (ribotyping)

Due to the high conservation of the rrn operon among prokaryotes, hybridisation studies

using 16S and 23S probes were frequently performed to give patterns usable for inter- and

intraspecific differentiation (70). The technique is however somewhat limited for Mycoplasma

spp., since the presence of only one or two copies of the rrn operon greatly decreases the

discriminatory power. As a consequence, mycoplasma-specific rrn probes were mainly used

for hybridisation experiments conducted directly on genomic DNA, without prior restriction

steps (see I.2.3.1). An important exception, however, is the pMC5 probe (1) containing the

5S, 23S and most of the 16S rRNA gene of M. capricolum. Initially, this probe was used to


detect mycoplasma contaminations of cell cultures, but was also shown useful to differentiate

strains (92, 219), also for bacterial species other than mycoplasmas (52).

I.2.2.2.2 With probes based on repetitive sequences

Probes targeting repeat regions are frequently used for typing purposes and may prove

especially valuable for mycoplasmas, for which an exceptional high fraction of the compact

genomes consist of repeated sequences (158). Even up to 29% of the genome of M. mycoides

subsp. mycoides SC consists of repetitive sequences, the highest density of all currently

completely sequenced bacteria (210). Generally, repeats are somewhat arbitrarily divided in

tandem repeated sequences (or satellites) and repeats that are dispersed around the genome.

These latter repeats are often linked to important surface antigens and, owing to occurring

recombination events between the multiple copies present, they may contribute to the

antigenic variation of mycoplasmas. Other dispersed repeated sequences show similarity to

known insertion sequences (IS) (116). In mycoplasmas, some IS have been well-described

(e.g. 25, 32, 144, 177, 211), while many others remain unassigned or have an unrevealed

mobile capacity. Therefore, the term IS-like elements (rather than IS) is typically used in

reference to these sequences. With the exponential increase of completely sequenced

genomes, many new such IS-like sequences became apparent. For instance, no less than four

different groups of IS-like elements were reported in the fully sequenced genome of M.

penetrans (160) and at least some of these may offer new possibilities for further

epidemiological studies. Indeed, most bacterial IS seem sufficiently stably integrated in the

genome to observe identical patterns after in vitro cultivation steps, although numerous

passages might result in slightly different IS profiles as was shown for at least M. bovis (177)

and M. fermentans (146). Overall, typing patterns based on the transposition of IS elements

allow to detect differences between strains over rather short periods of time (e.g. between two

subsequent outbreaks). Even for IS-like sequences without an inherent transposition activity,

the number and localisation in the genome usually differs between strains and may still be

used for short-term epidemiological studies. Therefore, DNA hybridisation studies using IS

sequences, called IS-fingerprinting, have frequently been used and proved highly

discriminative and reproducible (166).

Most IS-like elements are restricted to specific (subgroups of) species, although some are

present in different Mycoplasma spp. infecting the same host, pointing to events of horizontal

transmission. An IS-like element of M. mycoides subsp. mycoides SC, named ISMmy1, was

shown especially useful to differentiate the vaccine strain from field isolates. Several copies


of a nearly identical IS-like sequence were observed in M. bovis as well (177, 211). Likewise,

multiple copies of IS-like sequences, designated as IS1630 and IS1550 (ISMi1), have been

characterised in M. fermentans (25, 81), while similar sequences were detected in M. orale

(44). An IS-like sequence, characterised as IS1221, was shown to spread horizontally between

porcine mycoplasmas since several copies were detected in M. hyopneumoniae, M. hyorhinis,

and M. flocculare strains (57). The latter IS-like sequence may however not be functional

anymore because its transposase is most likely truncated (223). Another unassigned repetitive

sequence of M. hyopneumoniae carries long direct terminal repeats and is present in different

numbers in the genome (73). These sequcences may be useful for typing as well, but have

never been investigated thoroughly.

Other examples include IS-like elements that are (so far) only found in one species. Southern

hybridisation studies based on an IS-like element of M. mycoides subsp. mycoides SC, named

IS1296, lead to the conclusion that the re-emerging outbreaks in Europe involved a specific

clone, different from strains originating from Africa and Australia (32). More extensive

typing on African strains with a second DNA probe targeting IS1634 allowed further

differentiation between these strains (119). An IS-like element, named ISMag1 and which is

closely related to ISMbov1, was only found in rarely observed serogroups of M. agalactiae

(144). In this particular case, Southern hybridisation studies can only be used for these

subgroups, but may be of supplementary value to a conventionally used serological typing

method (13).

I.2.2.2.3 With probes based on other sequences

Nowadays, the sequence of the probe is used in order to determine its specificity, but at times

when sequence data were only sparsely available, the specificity was only determined

experimentally. In fact, DNA hybridisation studies can be performed with any ad random

genomic fragment provided that the specificity is adequate. Such a probe, named CAP-21,

comprising a ribosomal protein S7, was used to differentiate the closely related members of

the M. mycoides cluster (172) and also other probes have been used for the identification or

typing of several pathogenic mycoplasmas (62, 135, 183).


I.2.2.3 Based on restriction and PCR

I.2.2.3.1 Amplified Fragment Length Polymorphism (AFLP)

AFLP was originally developed to type plants, but the technique is also applicable for the

typing of bacteria (200). In short, genomic DNA is digested, typically using two restriction

enzymes. After (or during) restriction, double-stranded adapters are linked to the restriction

overhang-sites. Since adapters are constructed in such a way that the restriction recognition

sites are not restored after linkage, both restriction and linkage can be carried out

simultaneously. Subsequently, an amplification reaction is set up with primers complementary

to the ligated adapters. For most bacteria, but seldom for the small mycoplasmas, additional

selective bases are added to the 3’-end of the primers to generate on average 50 to 100

fragments that are separated using high-resolution electrophoresis. An exquisite protocol,

using a restriction step with BglII and MfeI and an amplification step without selective bases,

was optimised for the typing of several Mycoplasma spp. (96). Interestingly, the adapters of

this original AFLP protocol have overhangs on both sides (i.e. two different sticky ends), so

more restriction enzymes may be used. Later reports used the same protocol with only minor

modifications, such as the addition of a single adenosine to one of the primers for the typing

of avian species (80) or the use of restriction endonucleases EcoRI and Csp6I to differentiate

the members of the M. mycoides-cluster using identical adapters (99).

For mycoplasmas, the discriminatory power of AFLP was shown very high, mostly exceeding

that of other techniques such as PFGE (80, 96, 99, 127). AFLP was also demonstrated to be a

reproducible technique despite the minor differences in peak intensities common to all PCR

based techniques. The technique is also easier and faster to perform compared to PFGE, but

the analysis of the obtained fingerprints may be complex and needs sophisticated software.

AFLP studies carried out on mycoplasmas clearly showed the wide difference in the

variability of different species. Some Mycoplasma spp., like M. genitalium and M.

pneumoniae, showed a high degree of similarity, while others, like some porcine and avian

mycoplasmas, had widely diverse AFLP fingerprints (80, 96). For M. bovis strains originating

from the UK, two distinct clusters were apparent, despite a great intraspecific polymorphism

(127).


I.2.2.4 Based on PCR

I.2.2.4.1 Random Amplified Polymorphic DNA (RAPD) or Arbitrarily Primed PCR (AP-

PCR)

RAPD is a one-step, PCR-based typing technique using one short primer of about 10

nucleotides long, to amplify several genomic regions falling between two complementary

primer binding sites (206). Since the amplification reaction is carried out at a very low

annealing temperature, the stringency of the binding between primer and target sequence is

also low. As a consequence, the primer will bind abundantly on the genome. This key feature

of RAPD generally results in problems concerning reproducibility and even for reports

claiming nearly 100% reproducibility (121, 204), interpretation is complicated owing to

inconsistent band intensities. The slightest change in buffer conditions, DNA concentration or

temperature may yield different fingerprints. Since the course of the PCR cycle can slightly

differ between PCR apparatuses, interlaboratory comparison of RAPD patterns is often

difficult, if not impossible (138). Reproducibility can be improved by using pre-prepared

mastermixes, more stable polymerases such as Vent or Stoffel-fragment (63), or the use of

rather expensive, uniform RAPD-beads (Amersham Biosciences, Germany).

The success of the technique lays in its simplicity and speed. No knowledge about the genome

sequence is needed and the choice of good primers is simply a matter of trial and error,

although the discriminatory power of the method is greatly dependent on the primers chosen

(186). Although RAPD may also be useful to differentiate species (154), the technique is

especially suited for the differentiation of strains of the same species. Some RAPD primers

were even useful for the typing of strains of unrelated species, as shown for M. pneumoniae

and Ureaplasma urealyticum (36, 187). The high discriminatory index of RAPD, comparable

and sometimes even higher than that observed for PFGE, is an extra important benefit of the

technique. For mycoplasmas, another advantage of the technique is the high sensitivity of the

PCR reaction, such that only minute quantities of highly purified DNA are sufficient to obtain

a good fingerprint. Still, with other typing techniques at hand that favour interlaboratory

comparison, RAPD is unlikely to become the reference typing technique, despite the

standardised guidelines that have been proposed (155).

Many RAPD protocols have been described for mycoplasmas. Using the technique, a high

diversity was demonstrated between strains of many avian species (31, 53, 64, 93, 106, 121,

205), M. hyopneumoniae (2) and M. bovis (24, 127). Despite this heterogeneity of the latter


species (127), relatively stable RAPD patterns were observed for isolates originating from the

same farm over a six month period (24). Identical RAPD patterns of M. hominis strains were

also observed in women and their newborns (67), indicating the use of RAPD for the study of

vertical transmission. RAPD was also shown useful to display horizontal transfer since

similar RAPD patterns were observed for epidemiologically related strains of avian species

that are otherwise very heterogenic (31, 53, 64, 106).

I.2.2.4.2 Repetitive PCR

As an alternative to the arbitrary primer binding in RAPD, primers can be selected to amplify

regions between known repetitive sequences in an attempt to type strains. Since primers can

be chosen with a higher stringency, reproducibility will be less of a concern compared to

RAPD at least in theory (186). Such a 38 bp repetitive extragenic palindromic (rep) element

proved useful to type a whole range of prokaryotes, especially gram-negatives (198). Such a

similar repeat sequence has not been described for mycoplasmas. On the other hand, a

repetitive PCR based on a sequence from M. pneumoniae has been successfully used to type

Staphylococcus aureus strains (192).


I.2.3 Molecular techniques performed on defined chromosomal loci The number of published PCRs and hybridisation tests to correctly detect or identify

Mycoplasma spp. is overwhelming. Instead of providing a list of all these PCR tests, this

review will only focus on some general concepts and newer PCR technologies. As a

consequence, the presented data in these sections (I.2.3.1 and I.2.3.2) must be considered as

an update and summation of some other reviews on this issue (153, 156, 185).

I.2.3.1 Based on hybridisation

In contrary to cultivation and serological methods used for detection and subsequent

identification, hybridisation methods are generally more specific and faster. However, the

sensitivity may be lower than expected, making the method less attractive (153). Previously,

these methods were often used, but at present they have been largely replaced by PCR based

methods. An important exception however may be a technique called fluorescent in situ

hybridisation (FISH). Apart from the simultaneous detection and identification, FISH

provides additional information about the physical localisation of the probe target in a sample.

As a consequence, reports about the use of probes for the identification of mycoplasmas on

tissue sections are abundantly available (e.g. 21, 74, 87, 100).

I.2.3.2 Based on PCR

I.2.3.2.1 PCR

Ever since the invention in 1985, PCR has become one of the most frequently used techniques

for the rapid detection and identification of bacteria. The observed specificity, ease of

performance and sensitivity is so far unequalled in one single test. Although seemingly

perfect, the efficiency of PCR may, due to inhibitors, decrease dramatically when performed

directly on clinical samples. Several preprocessing methods may prove beneficial, but are by

no means generally applicable and DNA purification must be optimised for every type of

sample (150). A selective culture enrichment step followed by a short DNA purification step

is often used to avoid these problems, but may well reduce the sensitivity of PCR to the level

of conventional culture methods. Increased sensitivity may be acquired by the use of nested or

semi-nested PCRs, but care must be taken to reduce the risk of carry-over contamination,

giving false positive results (156). As long as erroneous results cannot be ruled out, PCR will

unlikely replace all conventional detection methods (195), although its value for identification

remains indisputable.


Various sorts of PCRs have been described to detect, identify or even type mycoplasmas.

Generally, the amplification products are visualised by standard gel electrophoresis, but also

more sophisticated separation techniques like high resolution or denaturing gradient gel

electrophoresis (DGGE) have been reported to resolve otherwise indistinguishable PCR

products. In addition, many identification and typing techniques make use of PCR in a first

step and the amplicons are subsequently used for hybridisation, restriction or sequence

analysis. This multitude of methods will be discussed below in greater detail.

(1) Single PCRs for the amplification of sequences conserved within a species

Many PCRs use 16S rRNA gene sequences as a target for the detection and identification of

Mycoplasma spp. (and bacteria in general) because of several reasons. First, 16S rRNA genes

are well conserved within a species (214). Secondly, the 16S rRNA gene sequences of almost

every known bacterium are available and the specificity of selected primers can be precisely

assessed in silico. Finally, 16S rRNA genes consist of highly conserved regions and regions

with higher interspecific variability. So, depending on the user’s needs, primers can be

selected to amplify entire groups or to differentiate single species. In case of mycoplasmas,

the latter assumption may not be completely correct for some very related species with nearly

identical 16S rRNA genes (103). Therefore, in order to differentiate 16S rRNA gene

amplification products of these very related species, different electrophoresis methods have

been proposed. Using high resolution electrophoresis, even a one base pair difference in

length of the rrnB of M. mycoides subsp. mycoides SC could be used to differentiate this

species from the other members of the M. mycoides cluster (139). DGGE was proven useful to

differentiate most Mycoplasma spp. on the basis of difference in their 16S rRNA gene

sequences using only one set of primers (126). Although this latter method was proven very

discriminative, many laboratories prefer to avoid the trouble of casting a gradient gel,

diminishing the success of the technique.

Alternatively to changes in electrophoresis conditions, other target sequences may be

preferable to distinguish Mycoplasma spp. Size differences between the amplification

products of the 16S-23S intergenic spacer (ITS) are used to differentiate many mycoplasmas

(e.g. 30, 170), including some very closely related species as M. gallisepticum and M. imitans

(76). Of course, also species-specific genes may be chosen as a target for amplification. Since

for most pathogenic mycoplasmas, genes involved in virulence are unknown, primers are

often selected on well-characterised adhesins or major lipoproteins. Repetitive DNA

sequences may be preferable targets since they may increase the sensitivity of the assay (203).


Outwardly directed tDNA primer sequences have also been proposed to differentiate species

(128, 207). The idea is that parts of tDNA sequences are highly conserved within prokaryotes

and consensus tDNA primers can be used in even distantly related bacteria. Within a genus,

tRNA genes are ordered within highly conserved cistrons, while the ITS between two

adjacent tRNA genes may be largely different. As a consequence, amplified ITS using

consensus primers followed by high-resolution electrophoresis for exact sizing may result in

species-specific patterns. Such high-resolution electrophoresis will not only improve the

accuracy of the technique, but also makes automation easier. The use of a sequencer results in

electronic datasets that can easily be shared amongst different laboratories or stored in online

databases (5). However, since electrophoresis is to a certain extent dependent on the

fluorochromes and the capillary used, peak profiles may not be exchangeable between groups

using slightly different protocols or equipment (39). The main advantage of the technique is

that one simple PCR can be used to identify most (if not all) species of a single genus. This

simple PCR method proved reproducible and discriminative for a number of bacterial species

(4, 6, 28, 40, 41, 104, 115, 196) and may be extended to the identification of Mycoplasma

spp. as well. With the choice of a correct set of primers, the technique may become a valuable

tool for the identification of numerous bacterial species (207). Although pure DNA samples

are preferred, mixed samples will not lead to misidentification and overlapping peak patterns

may occasionally even be resolved. Further studies are needed to determine whether the

technique also opens possibilities for an easy identification of the intracellular, plant-

pathogenic and uncultivable phytoplasmas. Although short intergenic distances are rarely

found in Eukarya (120), the presence of eukaryotic DNA may interfere owing to the presence

of tDNA-like sequences or cell-organelles containing tRNA genes (207).

(2) Single PCRs for the amplification of sequences that vary within a species

The majority of PCRs use target sequences that are conserved within a species to generate

fragments of known sizes, which are detected by electrophoresis. On the other hand, PCRs

amplifying polymorphic genes or reiterated repeat regions have been described to differentiate

strains or subgroups within a species. In fact, two different sorts of PCR can be distinguished.

A first kind of PCR uses different sets of primers, at which each set of primers will be specific

for a different subgroup of the species. In a second category, the PCR is performed with only

one set of primers, but the amplicon itself may differ in size depending on the strain under

investigation.


A PCR belonging to the first category has been described for M. pneumoniae. While many,

otherwise very useful, typing techniques seem to fail for this very homogeneous species, a

PCR, with a set of primers complementary to a variable region of the P1 gene, was shown

capable to subdivide strains into two distinct groups (50, 167). Apparently, M. pneumoniae

outbreaks alternate between these P1 subtypes. Since P1 functions as an adhesin, specific

antibodies of the host may block adherence and explain the observed shifts (49). Such

conclusions emphasise the mutual value of epidemiological and biological data.

Many more PCRs belonging to the second category have been described since hypervariable

regions or short tandem-repeat regions are abundantly present in Mycoplasma spp. (e.g. 18,

184, 213). Such PCRs can give a first idea about the existing variation between strains, but

results must be interpreted with care. The stability of the repeat regions must be checked to

ascertain that a PCR will not yield different amplicons for a same isolate over in vitro

passages. This is exemplified by the high-frequency rearrangements of the vsp genes, i.e. size-

variable surface lipoproteins of M. bovis, that were shown to be linked to phenotypic

switching after in vitro passages (113, 220) and similar rearrangements in vmpa genes may

affect M. agalactiae strains (58, 65). This continuous adaptation and phenotypic ON-OFF

switching is probably common to most mycoplasmas (47, 176) and may restrain the use of

some repetitive sequences for typing (189). Moreover, even if the stability is sufficiently high,

other techniques often prove superior for differentiating strains since the region under

examination is small and no conclusions can be made about relationships between strains

without at least sequencing the PCR product. Therefore, the epidemiological value is

generally limited, but the link with the biological importance of these genes in vivo certainly

adds to the value of the technique.

(3) Multiplex PCR

Aside from the numerous single PCR reactions available, a number of multiplex PCRs have

been developed for the simultaneous detection and/or identification of mycoplasmas that

reside in the same host (27, 33, 69, 165, 201). Other multiplex PCRs have been developed for

the detection of bacteria causing a multifactorial disease complex in which mycoplasmas may

be involved (117, 130, 137, 145). These PCRs largely reduce labour and costs, which is very

beneficial for diagnostic laboratories. On the other hand, multiplex PCRs are hard to optimise

and the conditions used are seldom perfect for all primer couples. As a result, their sensitivity

is in general lower than the separate single PCRs. In addition, when one species is abundantly

present, other species may remain undetected owing to substrate limitation (27). This may be


especially important in diseases where mycoplasmas are a primary agent and are quickly

outnumbered by secondary infections.

(4) Reverse transcriptase PCR (RT-PCR)

RT-PCR is a variation of the standard PCR technique in which cDNA is made from RNA via

reverse transcription. The cDNA is then amplified using standard PCR protocols. Owing to

the numerous copies of RNA present in viable cells, 1000 times higher sensitivities compared

to standard PCRs have been reported (193), although conflicting data exist (179). Since RNA

was reported to be stable up to about 23 hours (123), degradation of RNA or differences in

viable cells may partly explain the observed results. These difficulties in RNA extraction

compared to the far more stable DNA, makes the technique less interesting for many

applications. Still, a substantial number of RT-PCRs have been described for mycoplasmas

(e.g. 71, 123, 147, 179).

(5) Real-time PCR

Real-time PCRs are a recent breakthrough in PCR technology where the accumulation of

amplicons is measured during the reaction (215). The more templates present at the beginning

of the reaction, the fewer number of cycles it takes to reach a point in which the fluorescent

signal is recorded as statistically significant above the background signal. The sensitivity of

real-time PCRs is comparable to standard PCRs, but the collection of data as the reaction is

proceeding allows DNA quantitation. For mycoplasmas that cause chronic diseases and/or

remain present in healthy hosts for long periods of time, these quantitative data may prove

very beneficial for diagnosis. As a consequence, real-time PCR techniques to identify

Mycoplasma spp. are uprising, even in multiplex-format (Table 4).

I.2.3.2.2 PCR and hybridisation

In contrary to the many developed specific PCRs, unknown samples can be simultaneously

screened using an extensive number of different probes immobilised on a solid carrier.

Recently, reverse line blot hybridisation was described as a technique to detect and identify a

number of different Mollicutes spp. that commonly infect cell-cultures (202). In this method,

specimens were subjected to a nested-PCR to amplify the 16S-23S ITS. The labeled

amplicons were subsequently hybridised to species-specific probes fixed on a membrane

allowing the simultaneous identification of multiple species. Since the array technology is

booming, other applications and other platforms may be expected in the near future, even in

micro-array format.


Table 4: List of published real-time PCRs in mycoplasmology

Mycoplasma species Target sequence1

Sensitivity2, 3 References

M. gallisepticum MGA_0319 3 CFU (26) M. genitalium MgPa

gyrA 16S rDNA

< 5 copies < 10 copies < 5 copies

(85) (17) (89)

M. haemofelis 16S rDNA < 2 copies (171) M. hominis 16S rDNA

gap < 100 copies < 10 copies

(222) (3)

M. hyopneumoniae prl; MHP_580 < 10 copies (48) M. pneumoniae 16S rDNA

P1 16S rDNA P1 16S rDNA

5 CCU ND < 100 copies < 10 copies < 5 CFU

(108) (173) (90) (208) (151)

1 gap = glyceraldehyde-3-phosphate gene; MGA_0319 = conserved lipoprotein; MgPa = M. genitalium adhesin; gyrA = gyrase gene; prl = putative multidrug resistance protein gene; MHP_580 = hypothetical protein: repetitive region MHYP1-03-950; P1 = cytadhesin of M. pneumoniae.

2 ND = not determined; CCU = colour changing units; CFU = colony forming units. These numbers may be hard to compare since ‘copies’ denotes both live and dead bacteria, while CCU and CFU only takes into account live bacteria.

3 sensitivity is illustrated based on pure cultures or purified DNA. The sensitivity in clinical samples may be several magnitudes lower (26)

I.2.3.2.3 PCR and restriction

Locus specific amplification followed by restriction is a common method for the

identification and typing of mycoplasmas. Different names have been applied to the

technique, which may be found a bit confusing (194). Since the general term ‘restriction

fragment length polymorphism (RFLP)’ is often used for REA followed by DNA-

hybridisation, we will solely use the term PCR-RFLP. Whenever the technique is related to

rRNA genes, the term ‘amplified rDNA restriction analysis (ARDRA)’ may be preferable.

(1) For identification purposes

Many essential genes have orthologs in other species. Frequently, these genes contain both

regions that are conserved and regions that show more interspecific variability. As a

consequence, primers can be chosen on the conserved regions, while the more variable

regions can be used for species differentiation (and thus identification purposes). Restriction

analysis may be used to determine these sequence differences. Once the restriction enzymes

are selected, the technique is straightforward, making it possible to introduce it in any

molecular laboratory with basic equipment. Moreover, the discriminatory power of the


technique for the potentially useful restriction endonucleases can be estimated in silico as long

as the necessary sequences are available. Possible drawbacks of the technique are partial

restriction and intraspecific microheterogeneity that may complicate identification (78, 142).

Still, in most cases, microheterogeneity will lead to unknown restriction patterns rather than

to false identifications.

For mycoplasmas, a number of PCR-RFLP methods have already been described. Since

identification is still mostly based on 16S rRNA gene sequences (I.2.3.2.1.1), ARDRA was

already demonstrated to be useful for the identification of a number of Mycoplasma spp. (19,

37, 42, 54, 91, 139). Since high quality 16S rRNA gene sequences become more and more

available, the technique may rather easily be extended to differentiate other mycoplasmas as

well.

Apart from 16S rRNA gene sequences, a few reports stated the use of other sequences for the

identification of mycoplasmas using PCR-RFLP. The dnaK gene was proposed as a suitable

candidate to differentiate avian mycoplasmas (162), while also restriction of the 16S-23S ITS

region could be used to differentiate some closely related species (75). Recently,

amplification of a membrane-protein 81 gene followed by restriction analysis was described

for the rapid detection and differentiation of M. bovis and M. agalactiae (59).

(2) For typing purposes

PCR-RFLP may also be used to demonstrate differences between strains of the same species.

In fact, any variable gene or DNA fragment can be used for PCR-RFLP-analyses to visualise

differences, but the mutation rate or supplemental epidemiological data must be taken into

account before drawing definite conclusions. Since the locus under investigation is only a

minor fragment of the genome and may change abruptly, PCR-RFLP is not suited to visualise

relationships between strains. On the other hand, when the locus under investigation proves

sufficiently stable, PCR-RFLP may be a fast and accurate method to differentiate strains

and/or allocate them to certain well defined groups without the need of time-consuming

cultivation methods. Several examples where the technique was demonstrated to differentiate

mycoplasma strains have been reported. As discussed (I.2.3.2.1.2), differences in the P1

operon can be used to differentiate M. pneumoniae strains (36, 45). Furthermore, a single

nucleotide change in the bgl gene of highly virulent African M. mycoides subsp. mycoides

strains was an adequate marker to differentiate these strains from different geographical

origins (199). In another study, the variability of the pvpA gene was shown to be useful for

subdividing M. gallisepticum isolates according epidemiological outbreaks (107, 143).


I.2.3.2.4 PCR and sequence analysis

(1) Sequence analysis for identification

Determination of the 16S rRNA gene sequence is without any doubt one of the most direct

and accurate methods for the identification of bacteria, but is in general still rather expensive

to be used in routine diagnostics. Besides, the sequence of the entire gene may be needed for

some mycoplasmas that have nearly identical (>99%) 16S rRNA gene sequences (22).

Nevertheless, for human mycoplasmas and ureaplasmas, a semi-nested PCR on a conserved

part of the 16S rDNA followed by sequence analysis was reported as a generally applicable

and rapid method for identification (221).

(2) Sequence analysis for typing

(2.i) Based on single genomic fragments

While amplification of the species-specific PCR product can result in a direct identification,

sequence analysis can be used to discriminate between strains. The technique does not need

the cultivation of fastidious mycoplasmas and since all molecular typing techniques are

ultimately based on differences in sequences, sequence analysis seems the best approach.

Moreover, the technique has an excellent interlaboratory reproducibility and data can be

stored in online databases (88). Yet, sequence analysis of single genomic fragments has also

some important drawbacks. The region under investigation is very small and is hardly, if ever,

representative for the entire genome. Besides, the region under investigation must be

conserved enough for amplification and at the same time variable enough to differentiate

between strains, which is not always easily attainable. In addition, the stability of the gene

must be verified over in vitro passages. For most genes, however, these data are not available

and results must be interpreted with care, especially since hypervariable regions are not

uncommon in mycoplasmas. Even if the molecular clock of the particular genomic fragment

is known, intraspecific recombination events, as demonstrated to occur in a wide range of

bacterial species (23, 43, 83, 105, 110, 159), will be hard to detect and may lead to wrong

phylogenetic topologies. Finally, to avoid minor sequence errors, the sequence of both strands

should be determined at least once, preferably from two independent PCR reactions.

Naturally, this makes the technique very expensive.

Still, sequence analysis of species-specific genomic fragments have been proposed or

successfully used for the typing of some Mycoplasma spp. In case of M. genitalium, the

MG309 gene sequence was proven stable in sequential urine samples obtained from single

patients for at least five weeks and may be valuable candidates for further typing studies


(114). Another example of sequence analysis was demonstrated for a genomic fragment of

2400 bp of M. capricolum subp. capripneumoniae. Nucleotide variations in this specific

fragment were used to determine the geographical distribution of different strains (112).

Sequence variation of parts of the haemagglutinin encoding gene vlhA of M. synoviae was

used for strain differentiation (79) and could be linked to the length of the expressed protein

and to virulence (11).

(2.ii) Multi-locus sequence typing (MLST)

Although even more expensive (136), a technique called MLST was developed to cope with

some important drawbacks related to typing studies based on sequence analysis of single

genes. Instead of analyzing one single genomic fragment, the partial sequences of multiple,

selected genes are determined. Ideally, sequence fragments about 500 bp in length of at least

seven, widely scattered genes should be included to obtain a good representation of the

genome (29), but the number may vary between different studies. Mostly, essential

(housekeeping) genes are chosen because they are less often subjected to horizontal transfer

events and are not liable to strong or unusual selective pressures. As a consequence, they are

perfectly suited to represent the accumulation of sequence variation in the genome. However,

some exceptions have been reported where a number of housekeeping genes showed too few

differences to be useful for typing (50, 118, 134). For these cases, carefully selected species-

specific genes may be included instead. Once the base or amino acid substitution rate of the

selected genes is known, mathematical models are available to perform profound

phylogenetic analyses and to efficiently determine the clonal structure of the population (43,

83, 105, 159).

MLST turned out to be the method of choice for the typing of several bacterial species of

global importance (188) and for those, databases for international surveillance have been set

up (55). Only few investigators reported on the potential of the technique for Mycoplasma

spp. For the homogeneous M. pneumoniae strains, the housekeeping genes investigated

showed not sufficient differences and proved to be useless for MLST typing. Also the

sequences of repetitive regions or genes involved in cytadherence were surprisingly

homogeneous. Consequently, the discriminatory index of MLST was not shown to be superior

to many other techniques used for the typing of M. pneumoniae (46, 50). The potential of

MLST was also evaluated for M. mycoides subsp. mycoides SC. In agreement with Southern

hybridisation studies based on an IS-like element (I.2.2.2.2), partial sequences of four genes

sufficed to visualise the geographical distribution of clonal lineages (111). In still another


study, partial sequences of four species-specific genes were able to point to the strong

similarity of the pathogenic M. gallisepticum strains isolated from turkeys and a commonly

used 6/85 vaccine strain (93). Together with the amazing amount of variability observed in

the wild-type population of M. gallisepticum (56, 122), these data strongly suggested that the

vaccine strain may spread in nature. However, care must be taken with the interpretation of

these data, since it may be difficult to determine whether the virulent isolates are actually

derived from the vaccine strain or occur as a natural population amongst turkeys (93).

Table 5: Summary of the characteristics of various molecular typing techniques1 (adapted

from 124, 136, 190, 216).

Typing method2 Reproducibility

3 Discriminatory

Power

Ease of

Performance

Time required

(days)

Ease of

interpretation

Cost-

effective

REA +/- +/- ++ 1 -- ++

PFGE ++ ++ +/- 2-3 + +/-

Hybridisiation (ribotyping) ++ +/- +/- 2 + +

PCR-RFLP ++ +/- ++ 1 + +

RAPD - ++ ++ <1 +/- ++

AFLP + ++ +/- 2 - +/-

Sequence analysis (MLST) ++ ++ + 1-2 ++ - 1 different characteristics are cited from very high (++), over variable (+/-) to very low (--). However, it must be noted

that the given quotations are merely estimates and may vary depending on the species under investigation. 2 various typing techniques (spoligotyping, Rep-PCR, VNTR, … ) have not (yet) been described for mycoplasmas and

are consequently not included. 3 the term reproducibility is not to be confused with the stability of the typing data. Since both very homogeneous and

heterogeneous Mycoplasma spp. have been described, this latter term was left out of the table.


I.2.4 Future techniques & conclusion During the past decades, the importance of molecular techniques in mycoplasmology has

greatly increased. Still, no single technique seems to be perfect (Table 5). The discriminatory

power, applicability, reproducibility, ease of performance, and ease of interpretation, may

vary depending on the Mycoplasma species under investigation and must be evaluated for

each situation (136). The advancement of automation, which will become increasingly

important in the future, will likely help to further standardise actually available techniques

allowing improvement of the interlaboratory reproducibility. Even methods that are otherwise

considered outdated or too labour-intensive, may have potential use in a fully automated

format. For instance, a DNA-hybridisation technique with rDNA probes yielded reproducible

fingerprints useful for identification (RiboPrinter, Dupont, De, USA). On the other hand,

newer techniques are constantly developed and may offer new possibilities. The current

expansion in biosensors and microchip evolution may become an affordable and generally

applicable alternative to many current identification and typing techniques. Also the

continuous progress on the determination of whole genome sequences may lead to additional

targets and/or repetitive sequences for the development of new typing methods. Typing

systems based on variable number of tandem-repeats (VNTR) have already been proposed for

Mycobacterium tuberculosis, Salmonella enterica, and Neisseria meningitidis (152, 168, 217)

and related systems might be developed for some Mycoplasma spp. as well.

This progress in molecular techniques will lead to faster and easier attainable methods to

detect and identify mycoplasmas or even to point to the existence of new species.

Furthermore, the increase in epidemiological knowledge will help to elucidate the prevalence

and geographical spreading of mycoplasmas over time or the extent and mode of transmission

of clones during outbreaks. One day it may not be enough to describe which species, but

rather which specific strain is implicated in disease as ever more reports link differences

between strains to differences observed in biological properties.


References

1. Amikam, D., S. Razin, and G. Glaser. 1982. Ribosomal RNA genes in mycoplasma. Nucleic Acids

Res. 10:4215-4222.

2. Artiushin, S., and F. C. Minion. 1996. Arbitrarily primed PCR analysis of Mycoplasma

hyopneumoniae field isolates demonstrates genetic heterogeneity. Int. J. Syst. Bacteriol. 46:324-328.

3. Baczynska, A., H. F. Svenstrup, J. Fedder, S. Birkelund, and G. Christiansen. 2004. Development

of real-time PCR for detection of Mycoplasma hominis. BMC Microbiol. 4:35.

4. Baele, M., P. Baele, M. Vaneechoutte, V. Storms, P. Butaye, L. A. Devriese, G. Verschraegen, M.

Gillis, and F. Haesebrouck. 2000. Application of tRNA intergenic spacer PCR for identification of

Enterococcus species. J. Clin. Microbiol. 38:4201-4207.

5. Baele, M., V. Storms, F. Haesebrouck, L. A. Devriese, M. Gillis, G. Verschraegen, T. de Baere,

and M. Vaneechoutte. 2001. Application and evaluation of the interlaboratory reproducibility of tRNA

intergenic length polymorphism analysis (tDNA-PCR) for identification of Streptococcus species. J.

Clin. Microbiol. 39:1436-1442.

6. Baele, M., M. Vaneechoutte, R. Verhelst, M. Vancanneyt, L. A. Devriese, and F. Haesebrouck.

2002. Identification of Lactobacillus species using tDNA-PCR. J. Microbiol. Methods. 50:263-271.

7. Barlev, N. A., and S. N. Borchsenius. 1991. Continuous distribution of mycoplasma genome sizes.

Biomed. Sci. 2:641-645.

8. Bautsch, W. 1988. Rapid physical mapping of the Mycoplasma mobile genome by two-dimensional

field inversion gel electrophoresis techniques. Nucleic Acids Res. 16:11461-11467.

9. Bebear, C. M., O. Grau, A. Charron, H. Renaudin, D. Gruson, and C. Bebear. 2000. Cloning and

nucleotide sequence of the DNA gyrase (gyrA) gene from Mycoplasma hominis and characterization of

quinolone-resistant mutants selected in vitro with trovafloxacin. Antimicrob Agents Chemother.

44:2719-2727.

10. Behrens, A., F. Poumarat, D. L. Grand, M. Heller, and R. Rosengarten. 1996. A newly identified

immunodominant membrane protein (pMB67) involved in Mycoplasma bovis surface antigenic

variation. microbiology. 142:2463-2470.

11. Bencina, D., and J. M. Bradbury. 1992. Combination of immunofluorescence and immunoperoxidase

techniques for serotyping mixtures of Mycoplasma species. J. Clin. Microbiol. 30:407-410.

12. Berent, L. M., and J. B. Messick. 2003. Physical map and genome sequencing survey of Mycoplasma

haemofelis (Haemobartonella felis). Infect. Immun. 71:3657-3662.


13. Bergonier, D., F. De Simone, P. Russo, M. Solsona, M. Lambert, and F. Poumarat. 1996. Variable

expression and geographic distribution of Mycoplasma agalactiae surface epitopes demonstrated with

monoclonal antibodies. FEMS Microbiol. Lett. 143:159-165.

14. Bernal, A., U. Ear, and N. Kyrpides. 2001. Genomes OnLine Database (GOLD): a monitor of

genome projects world-wide. Nucleic Acids Res. 29:126-127.

15. Blank, W. A., and G. W. Stemke. 2001. A physical and genetic map of the Mycoplasma flocculare

ATCC 27716 chromosome reveals large genomic inversions when compared with that of Mycoplasma

hyopneumoniae strain J(T). Int. J. Syst. Evol. Microbiol. 51:1395-1399.

16. Blank, W. A., and G. W. Stemke. 2000. A physical and genetic map of the Mycoplasma

hyopneumoniae strain J genome. Can. J. Microbiol. 46:832-840.

17. Blaylock, M. W., O. Musatovova, J. G. Baseman, and J. B. Baseman. 2004. Determination of

infectious load of Mycoplasma genitalium in clinical samples of human vaginal cells. J. Clin.


18. Boesen, T., J. Emmersen, A. Baczynska, S. Birkelund, and G. Christiansen. 2004. The vaa locus of

Mycoplasma hominis contains a divergent genetic islet encoding a putative membrane protein. BMC

Microbiol. 4:37.

19. Bolske, G., J. G. Mattsson, C. R. Bascunana, K. Bergstrom, H. Wesonga, and K. E. Johansson.

1996. Diagnosis of contagious caprine pleuropneumonia by detection and identification of Mycoplasma

capricolum subsp. capripneumoniae by PCR and restriction enzyme analysis. J Clin Microbiol. 34:785-

791.

20. Bové, J. M., and C. Saillard. 1979. Cell biology of spiroplasmas, p. 85-154. In M. F. Barile, S. Razin,

R. F. Whitcomb, and J. G. Tully (ed.), The mycoplasmas: plant and insects mycoplasmas, vol. III.

Academic Press, Inc., London.

21. Boye, M., T. K. Jensen, P. Ahrens, T. Hagedorn-Olsen, and N. F. Friis. 2001. In situ hybridisation

for identification and differentiation of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae and

Mycoplasma hyorhinis in formalin-fixed porcine tissue sections. APMIS. 109:656-664.



23. Brown, E. W., M. K. Mammel, J. E. LeClerc, and T. A. Cebula. 2003. Limited boundaries for

extensive horizontal gene transfer among Salmonella pathogens. Proc. Natl. Acad. Sci. USA.

100:15676-15681.

24. Butler, J. A., C. C. Pinnow, J. U. Thomson, S. Levisohn, and R. F. Rosenbusch. 2001. Use of

arbitrarily primed polymerase chain reaction to investigate Mycoplasma bovis outbreaks. Vet.



25. Calcutt, M. J., J. L. Lavrrar, and K. S. Wise. 1999. IS1630 of Mycoplasma fermentans, a novel IS30-

type insertion element that targets and duplicates inverted repeats of variable length and sequence

during insertion. J. Bacteriol. 181:7597-7607.

26. Carli, K. T., and A. Eyigor. 2003. Real-time polymerase chain reaction for Mycoplasma gallisepticum

in chicken trachea. Avian Dis. 47:712-717.

27. Caron, J., M. Ouardani, and S. Dea. 2000. Diagnosis and differentiation of Mycoplasma

hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR and amplification of the p36 and

p46 genes. J. Clin. Microbiol. 38:1390-1396.

28. Catry, B., M. Baele, G. Opsomer, A. de Kruif, A. Decostere, and F. Haesebrouck. 2004. tRNA-

intergenic spacer PCR for the identification of Pasteurella and Mannheimia spp. Vet. Microbiol.

98:251-260.

29. Caugant, D. A. 2001. From multilocus enzyme electrophoresis to multilocus sequence typing, p. 299-

349. In L. Dijkshoorn, K. J. Towner, and M. Struelens (ed.), New approaches for the generation and

analysis of microbial typing data. Elsevier B.V., Amsterdam, The Netherlands.

30. Chalker, V. J., W. M. Owen, C. J. Paterson, and J. Brownlie. 2004. Development of a polymerase

chain reaction for the detection of Mycoplasma felis in domestic cats. Vet. Microbiol. 100:77-82.

31. Charlton, B. R., A. A. Bickford, R. L. Walker, and R. Yamamoto. 1999. Complementary randomly

amplified polymorphic DNA (RAPD) analysis patterns and primer sets to differentiate Mycoplasma

gallisepticum strains. J. Vet. Diagn. Invest. 11:158-161.

32. Cheng, X., J. Nicolet, F. Poumarat, J. Regalla, F. Thiaucourt, and J. Frey. 1995. Insertion element

IS1296 in Mycoplasma mycoides subsp. mycoides small colony identifies a European clonal line

distinct from African and Australian strains. Microbiology. 141:3221-3228.

33. Choppa, P. C., A. Vojdani, C. Tagle, R. Andrin, and L. Magtoto. 1998. Multiplex PCR for the

detection of Mycoplasma fermentans, M. hominis and M. penetrans in cell cultures and blood samples

of patients with chronic fatigue syndrome. Mol. Cell. Probes. 12:301-308.

34. Cohan, F. M. 2002. What are bacterial species? Annu. Rev. Microbiol. 56:457-487.

35. Colman, S. D., P. C. Hu, W. Litaker, and K. F. Bott. 1990. A physical map of the Mycoplasma

genitalium genome. Mol. Microbiol. 4:683-687.

36. Cousin-Allery, A., A. Charron, B. de Barbeyrac, G. Fremy, J. Skov Jensen, H. Renaudin, and C.

Bebear. 2000. Molecular typing of Mycoplasma pneumoniae strains by PCR-based methods and

pulsed-field gel electrophoresis. Application to French and Danish isolates. Epidemiol. Infect. 124:103-

111.

37. Criado-Fornelio, A., A. Martinez-Marcos, A. Buling-Sarana, and J. C. Barba-Carretero. 2003.

Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from

southern Europe: a molecular study. Vet. Microbiol. 93:307-317.


38. Davis, M. A., D. D. Hancock, T. E. Besser, and D. R. Call. 2003. Evaluation of pulsed-field gel

electrophoresis as a tool for determining the degree of genetic relatedness between strains of

Escherichia coli O157:H7. J. Clin. Microbiol. 41:1843-1849.

39. De Baere, T., A. Van Keerberghen, P. Van Hauwe, H. De Beenhouwer, A. Boel, G. Verschraegen,

G. Claeys, and M. Vaneechoutte. 2005. An interlaboratory comparison of ITS2-PCR for the

identification of yeasts, using the ABI Prism 310 and CEQ8000 capillary electrophoresis systems. BMC

Microbiol. 5:14.

40. De Gheldre, Y., N. Maes, F. L. Presti, J. Etienne, and M. Struelens. 2001. Rapid identification of

clinically relevant Legionella spp. by analysis of transfer DNA intergenic spacer length polymorphism.

J. Clin. Microbiol. 39:162-169.

41. De Gheldre, Y., P. Vandamme, H. Goossens, and M. J. Struelens. 1999. Identification of clinically

relevant viridans streptococci by analysis of transfer DNA intergenic spacer length polymorphism. Int.

J. Syst. Bacteriol. 49 Pt 4:1591-1598.

42. Deng, S., C. Hiruki, J. A. Robertson, and G. W. Stemke. 1992. Detection by PCR and differentiation

by restriction fragment length polymorphism of Acholeplasma, Spiroplasma, Mycoplasma, and

Ureaplasma, based upon 16S rRNA genes. PCR Methods Appl. 1:202-204.

43. Dingle, K. E., F. M. Colles, D. R. Wareing, R. Ure, A. J. Fox, F. E. Bolton, H. J. Bootsma, R. J.

Willems, R. Urwin, and M. C. Maiden. 2001. Multilocus sequence typing system for Campylobacter

jejuni. J. Clin. Microbiol. 39:14-23.

44. Ditty, S. E., M. A. Connolly, B. J. Li, and S. C. Lo. 1999. Mycoplasma orale has a sequence similar

to the insertion-like sequence of M. fermentans. Mol. Cell. Probes. 13:183-189.

45. Dorigo-Zetsma, J. W., J. Dankert, and S. A. Zaat. 2000. Genotyping of Mycoplasma pneumoniae

clinical isolates reveals eight P1 subtypes within two genomic groups. J. Clin. Microbiol. 38:965-970.

46. Dorigo-Zetsma, J. W., B. Wilbrink, J. Dankert, and S. A. Zaat. 2001. Mycoplasma pneumoniae P1

type 1- and type 2-specific sequences within the P1 cytadhesin gene of individual strains. Infect.

Immun. 69:5612-5618.

47. Droesse, M., G. Tangen, I. Gummelt, H. Kirchhoff, L. R. Washburn, and R. Rosengarten. 1995.

Major membrane proteins and lipoproteins as highly variable immunogenic surface components and

strain-specific antigenic markers of Mycoplasma arthritidis. Microbiology. 141 ( Pt 12):3207-3219.

48. Dubosson, C. R., C. Conzelmann, R. Miserez, P. Boerlin, J. Frey, W. Zimmermann, H. Hani, and

P. Kuhnert. 2004. Development of two real-time PCR assays for the detection of Mycoplasma

hyopneumoniae in clinical samples. Vet. Microbiol. 102:55-65.

49. Dumke, R., I. Catrein, R. Herrmann, and E. Jacobs. 2004. Preference, adaptation and survival of

Mycoplasma pneumoniae subtypes in an animal model. Int. J. Med. Microbiol. 294:149-155.


50. Dumke, R., I. Catrein, E. Pirkil, R. Herrmann, and E. Jacobs. 2003. Subtyping of Mycoplasma

pneumoniae isolates based on extended genome sequencing and on expression profiles. Int. J. Med.

Microbiol. 292:513-525.

51. Dybvig, K., and L. L. Voelker. 1996. Molecular biology of mycoplasmas. Annu Rev Microbiol.

50:25-57.

52. Faibra, D. T. 1993. Heterogeneity among Dermatophilus congolensis isolates demonstrated by

restriction fragment length polymorphisms. Rev. Elev. Med. Vet. Pays. Trop. 46:253-256.

53. Fan, H. H., S. H. Kleven, and M. W. Jackwood. 1995. Studies of intraspecies heterogeneity of

Mycoplasma synoviae, M. meleagridis, and M. iowae with arbitrarily primed polymerase chain reaction.

Avian Dis. 39:766-777.

54. Fan, H. H., S. H. Kleven, M. W. Jackwood, K. E. Johansson, B. Pettersson, and S. Levisohn. 1995.

Species identification of avian mycoplasmas by polymerase chain reaction and restriction fragment

length polymorphism analysis. Avian Dis. 39:398-407.

55. Feil, E. J., and M. C. Enright. 2004. Analyses of clonality and the evolution of bacterial pathogens.

Curr. Opin. Microbiol. 7:308-313.

56. Ferraz, N. P., M. das Graças, and M. Danelli. 2003. Phenotypic and antigenic variation of

Mycoplasma gallisepticum vaccine strains. Braz. J. Microbiol. 34:238-241.

57. Ferrell, R. V., M. B. Heidari, K. S. Wise, and M. A. McIntosh. 1989. A mycoplasma genetic element

resembling prokaryotic insertion sequences. Mol. Microbiol. 3:957-967.

58. Flitman-Tene, R., S. Levisohn, I. Lysnyansky, E. Rapoport, and D. Yogev. 2000. A chromosomal

region of Mycoplasma agalactiae containing vsp-related genes undergoes in vivo rearrangement in

naturally infected animals. FEMS Microbiol. Lett. 191:205-212.

59. Foddai, A., G. Idini, M. Fusco, N. Rosa, C. de la Fe, S. Zinellu, L. Corona, and S. Tola. 2005.

Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-

PCR and a PCR-RFLP. Mol. Cell. Probes. 19:207-212.

60. Forbes, K. J., K. D. Bruce, J. Z. Jordens, A. Ball, and T. H. Pennington. 1991. Rapid methods in

bacterial DNA fingerprinting. J. Gen. Microbiol. 137:2051-2058.

61. Frey, J. 1998. Insertion sequence analysis. Methods Mol. Biol. 104:197-205.

62. Fujimoto, S., K. Umene, M. Saito, K. Horikawa, and M. J. Blaser. 2000. Restriction fragment length

polymorphism analysis using random chromosomal gene probes for epidemiological analysis of

Campylobacter jejuni infections. J. Clin. Microbiol. 38:1664-1667.

63. Geary, S. J., and M. H. Forsyth. 1996. PCR: Random amplified polymorphic DNA fingerprinting, p.

25-75. In J. G. Tully, and S. Razin (ed.), Molecular and Diagnostic procedures in mycoplasmology:

Diagnostic Procedures, vol. II. Academic Press Inc., San Diego.


64. Geary, S. J., M. H. Forsyth, S. Aboul Saoud, G. Wang, D. E. Berg, and C. M. Berg. 1994.

Mycoplasma gallisepticum strain differentiation by arbitrary primer PCR (RAPD) fingerprinting. Mol.

Cell. Probes. 8:311-316.

65. Glew, M. D., L. Papazisi, F. Poumarat, D. Bergonier, R. Rosengarten, and C. Citti. 2000.

Characterization of a multigene family undergoing high-frequency DNA rearrangements and coding for

abundant variable surface proteins in Mycoplasma agalactiae. Infect. Immun. 68:4539-4548.

66. Gorton, T. S., M. S. Goh, and S. J. Geary. 1995. Physical mapping of the Mycoplasma gallisepticum

S6 genome with localization of selected genes. J. Bacteriol. 177:259-263.

67. Grattard, F., B. Soleihac, B. De Barbeyrac, C. Bebear, P. Seffert, and B. Pozzetto. 1995.

Epidemiologic and molecular investigations of genital mycoplasmas from women and neonates at

delivery. Pediatr. Infect. Dis. J. 14:853-858.

68. Grau, O., F. Laigret, P. Carle, J. G. Tully, D. L. Rose, and J. M. Bove. 1991. Identification of a

plant-derived mollicute as a strain of an avian pathogen, Mycoplasma iowae, and its implications for

mollicute taxonomy. Int. J. Syst. Bacteriol. 41:473-478.

69. Greco, G., M. Corrente, V. Martella, A. Pratelli, and D. Buonavoglia. 2001. A multiplex-PCR for

the diagnosis of contagious agalactia of sheep and goats. Mol. Cell. Probes. 15:21-25.

70. Grimont, F., and P. A. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as

potential taxonomic tools. Ann. Inst. Pasteur Microbiol. 137B:165-175.

71. Grondahl, B., W. Puppe, A. Hoppe, I. Kuhne, J. A. Weigl, and H. J. Schmitt. 1999. Rapid

identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex

reverse transcription-PCR: feasibility study. J. Clin. Microbiol. 37:1-7.

72. Gurrieri, S., S. B. Smith, K. S. Wells, I. D. Johnson, and C. Bustamante. 1996. Real-time imaging

of the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees and 120 degrees

pulsed field gel electrophoresis. Nucleic Acids Res. 24:4759-4767.

73. Harasawa, R., K. Asada, and I. Kato. 1995. A novel repetitive sequence from Mycoplasma

hyopneumoniae. J. Vet. Med. Sci. 57:557-558.

74. Harasawa, R., K. Koshimizu, O. Takeda, T. Uemori, K. Asada, and I. Kato. 1991. Detection of

Mycoplasma hyopneumoniae DNA by the polymerase chain reaction. Mol. Cell. Probes. 5:103-109.

75. Harasawa, R., H. Mizusawa, K. Nozawa, T. Nakagawa, K. Asada, and I. Kato. 1993. Detection and

tentative identification of dominant Mycoplasma species in cell cultures by restriction analysis of the

16S-23S rRNA intergenic spacer regions. Res. Microbiol. 144:489-493.

76. Harasawa, R., D. G. Pitcher, A. S. Ramirez, and J. M. Bradbury. 2004. A putative transposase gene

in the 16S-23S rRNA intergenic spacer region of Mycoplasma imitans. Microbiology. 150:1023-1029.


77. Hege, R., W. Zimmermann, R. Scheidegger, and K. D. Stärk. 2002. Incidence of reinfections with

Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae in pig farms located in respiratory-

disease-free regions of Switzerland--identification and quantification of risk factors. Acta Vet. Scand.

43:145-156.

78. Heldtander, M., B. Pettersson, J. G. Tully, and K. E. Johansson. 1998. Sequences of the 16S rRNA

genes and phylogeny of the goat mycoplasmas Mycoplasma adleri, Mycoplasma auris, Mycoplasma

cottewii and Mycoplasma yeatsii. Int. J. Syst. Bacteriol. 48 Pt 1:263-268.

79. Hong, Y., M. Garcia, V. Leiting, D. Bencina, L. Dufour-Zavala, G. Zavala, and S. H. Kleven.

2004. Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA

sequence analysis targeting the hemagglutinin encoding gene vlhA. Avian Dis. 48:606-616.

80. Hong, Y., M. Garcia, S. Levisohn, I. Lysnyansky, V. Leiting, P. H. Savelkoul, and S. H. Kleven.

2005. Evaluation of amplified fragment length polymorphism for differentiation of avian Mycoplasma

species. J. Clin. Microbiol. 43:909-912.

81. Hu, W. S., R. Y. Wang, R. S. Liou, J. W. Shih, and S. C. Lo. 1990. Identification of an insertion-

sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus. Gene.

93:67-72.

82. Ionas, G., N. G. Norman, J. K. Clarke, and R. B. Marshall. 1991. A study of the heterogeneity of

isolates of Mycoplasma ovipneumoniae from sheep in New Zealand. Vet. Microbiol. 29:339-347.

83. Iredell, J., D. Blanckenberg, M. Arvand, S. Grauling, E. J. Feil, and R. J. Birtles. 2003.

Characterization of the natural population of Bartonella henselae by multilocus sequence typing. J.


84. Isaac, P. G., J. Stacey, and C. M. Clee. 1995. Nonradioactive probes. Mol. Biotechnol. 3:259-265.

85. Jensen, J. S., E. Bjornelius, B. Dohn, and P. Lidbrink. 2004. Use of TaqMan 5' nuclease real-time

PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis

who were attendees at a sexually transmitted disease clinic. J. Clin. Microbiol. 42:683-692.

86. Jensen, L. T., P. Thorsen, B. Moller, S. Birkelund, and G. Christiansen. 1998. Antigenic and

genomic homogeneity of successive Mycoplasma hominis isolates. J. Med. Microbiol. 47:659-666.

87. Johansson, K. E., J. G. Mattsson, K. Jacobsson, C. Fernandez, K. Bergstrom, G. Bolske, P.

Wallgren, and U. B. Gobel. 1992. Specificity of oligonucleotide probes complementary to

evolutionarily variable regions of 16S rRNA from Mycoplasma hyopneumoniae and Mycoplasma

hyorhinis. Res. Vet. Sci. 52:195-204.

88. Jolley, K. A., M. S. Chan, and M. C. Maiden. 2004. mlstdbNet - distributed multi-locus sequence

typing (MLST) databases. BMC Bioinformatics. 5:86.


89. Jurstrand, M., J. S. Jensen, H. Fredlund, L. Falk, and P. Molling. 2005. Detection of Mycoplasma

genitalium in urogenital specimens by real-time PCR and by conventional PCR assay. J. Med.


90. Khanna, M., J. Fan, K. Pehler-Harrington, C. Waters, P. Douglass, J. Stallock, S. Kehl, and K. J.

Henrickson. 2005. The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows

simultaneous detection of five organisms, Mycoplasma pneumoniae, Chlamydia (Chlamydophila)

pneumoniae, Legionella pneumophila, Legionella micdadei, and Bordetella pertussis, and its real-time

counterpart. J Clin Microbiol. 43:565-571.

91. Kiss, I., K. Matiz, E. Kaszanyitzky, Y. Chavez, and K. E. Johansson. 1997. Detection and

identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length

polymorphism assay. Vet. Microbiol. 58:23-30.

92. Kleven, S. H., G. F. Browning, D. M. Bulach, E. Ghiocas, C. J. Morrow, and K. G. Whithear.

1988. Examination of Mycoplasma gallisepticum strains using restriction endonuclease DNA analysis

and DNA-DNA hybridisation. Avian pathology. 17:559-570.

93. Kleven, S. H., R. M. Fulton, M. Garcia, V. N. Ikuta, V. A. Leiting, T. Liu, D. H. Ley, K. N.

Opengart, G. N. Rowland, and E. Wallner-Pendleton. 2004. Molecular characterization of

Mycoplasma gallisepticum isolates from turkeys. Avian Dis. 48:562-569.

94. Kleven, S. H., C. J. Morrow, and K. G. Whithear. 1988. Comparison of Mycoplasma gallisepticum

strains by hemagglutination- inhibition and restriction endonuclease analysis. Avian Dis. 32:731-741.

95. Kokotovic, B., N. F. Friis, and P. Ahrens. 2002. Characterization of Mycoplasma hyosynoviae strains

by amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis and 16S

ribosomal DNA sequencing. J. Vet. Med. B. Infect. Dis. Vet. Public Health. 49:245-252.

96. Kokotovic, B., N. F. Friis, J. S. Jensen, and P. Ahrens. 1999. Amplified-fragment length

polymorphism fingerprinting of Mycoplasma species. J. Clin. Microbiol. 37:3300-3307.

97. Krause, D. C., and C. B. Mawn. 1990. Physical analysis and mapping of the Mycoplasma pneumoniae

chromosome. J. Bacteriol. 172:4790-4797.

98. Kusiluka, L. J., B. Ojeniyi, and N. F. Friis. 2000. Increasing prevalence of Mycoplasma bovis in

Danish cattle. Acta Vet. Scand. 41:139-146.

99. Kusiluka, L. J., B. Ojeniyi, N. F. Friis, B. Kokotovic, and P. Ahrens. 2001. Molecular analysis of

field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides

subspecies mycoides, small colony type isolated from goats in Tanzania. Vet. Microbiol. 82:27-37.

100. Kwon, D., and C. Chae. 1999. Detection and localization of Mycoplasma hyopneumoniae DNA in

lungs from naturally infected pigs by in situ hybridization using a digoxigenin-labeled probe. Vet.

Pathol. 36:308-313.


101. Ladefoged, S. A., and G. Christiansen. 1998. Mycoplasma hominis expresses two variants of a cell-

surface protein, one a lipoprotein, and one not. Microbiology. 144 ( Pt 3):761-770.

102. Ladefoged, S. A., and G. Christiansen. 1992. Physical and genetic mapping of the genomes of five

Mycoplasma hominis strains by pulsed-field gel electrophoresis. J. Bacteriol. 174:2199-2207.

103. Le Grand, D., E. Saras, D. Blond, M. Solsona, and F. Poumarat. 2004. Assessment of PCR for

routine identification of species of the Mycoplasma mycoides cluster in ruminants. Vet. Res. 35:635-

649.

104. Lee, M. K., and A. J. Park. 2001. Rapid species identification of coagulase negative staphylococci by

rRNA spacer length polymorphism analysis. J Infect. 42:189-194.

105. Lemee, L., A. Dhalluin, M. Pestel-Caron, J. F. Lemeland, and J. L. Pons. 2004. Multilocus

sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types.


106. Ley, D. H., J. E. Berkhoff, and S. Levisohn. 1997. Molecular epidemiologic investigations of

Mycoplasma gallisepticum conjunctivitis in songbirds by random amplified polymorphic DNA

analyses. Emerg. Infect. Dis. 3:375-380.

107. Liu, T., M. Garcia, S. Levisohn, D. Yogev, and S. H. Kleven. 2001. Molecular variability of the

adhesin-encoding gene pvpA among Mycoplasma gallisepticum strains and its application in diagnosis.


108. Loens, K., M. Ieven, D. Ursi, T. Beck, M. Overdijk, P. Sillekens, and H. Goossens. 2003. Detection

of Mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification. J. Clin. Microbiol.

41:4448-4850.

109. Loens, K., D. Ursi, H. Goossens, and M. Ieven. 2003. Molecular diagnosis of Mycoplasma

pneumoniae respiratory tract infections. J. Clin. Microbiol. 41:4915-4923.

110. Lorenz, M. G., and J. Sikorski. 2000. The potential for intraspecific horizontal gene exchange by

natural genetic transformation: sexual isolation among genomovars of Pseudomonas stutzeri.

Microbiology. 146 Pt 12:3081-3090.

111. Lorenzon, S., I. Arzul, A. Peyraud, P. Hendrikx, and F. Thiaucourt. 2003. Molecular epidemiology

of contagious bovine pleuropneumonia by multilocus sequence analysis of Mycoplasma mycoides

subspecies mycoides biotype SC strains. Vet. Microbiol. 93:319-333.

112. Lorenzon, S., H. Wesonga, L. Ygesu, T. Tekleghiorgis, Y. Maikano, M. Angaya, P. Hendrikx, and

F. Thiaucourt. 2002. Genetic evolution of Mycoplasma capricolum subsp. capripneumoniae strains

and molecular epidemiology of contagious caprine pleuropneumonia by sequencing of locus H2. Vet



113. Lysnyansky, I., R. Rosengarten, and D. Yogev. 1996. Phenotypic switching of variable surface

lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. J. Bacteriol.

178:5395-5401.

114. Ma, L., and D. H. Martin. 2004. Single-Nucleotide polymorphisms in the rRNA operon and variable

numbers of tandem repeats in the lipoprotein gene among Mycoplasma genitalium strains from clinical

specimens. J. Clin. Microbiol. 42:4876-4888.

115. Maes, N., Y. De Gheldre, R. De Ryck, M. Vaneechoutte, H. Meugnier, J. Etienne, and M. J.

Struelens. 1997. Rapid and accurate identification of Staphylococcus species by tRNA intergenic

spacer length polymorphism analysis. J. Clin. Microbiol. 35:2477-2481.

116. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol. Mol. Biol. Rev. 62:725-774.

117. Mahony, J. B., D. Jang, S. Chong, K. Luinstra, J. Sellors, M. Tyndall, and M. Chernesky. 1997.

Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, and

Mycoplasma genitalium in first-void urine Specimens by multiplex polymerase chain reaction. Mol.

Diagn. 2:161-168.

118. Manning, G., C. G. Dowson, M. C. Bagnall, I. H. Ahmed, M. West, and D. G. Newell. 2003.

Multilocus sequence typing for comparison of veterinary and human isolates of Campylobacter jejuni.

Appl. Environ. Microbiol. 69:6370-6379.

119. March, J. B., J. Clark, and M. Brodlie. 2000. Characterization of strains of Mycoplasma mycoides

subsp. mycoides small colony type isolated from recent outbreaks of contagious bovine

pleuropneumonia in Botswana and Tanzania: evidence for a new biotype. J. Clin. Microbiol. 38:1419-

1425.

120. Marck, C., and H. Grosjean. 2002. tRNomics: analysis of tRNA genes from 50 genomes of Eukarya,

Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features. RNA 8:1189-

1232.

121. Marois, C., F. Dufour-Gesbert, and I. Kempf. 2001. Comparison of pulsed-field gel electrophoresis

with random amplified polymorphic DNA for typing of Mycoplasma synoviae. Vet. Microbiol. 79:1-9.

122. Marois, C., F. Dufour-Gesbert, and I. Kempf. 2001. Molecular differentiation of Mycoplasma

gallisepticum and Mycoplasma imitans strains by pulsed-field gel electrophoresis and random amplified

polymorphic DNA. J. Vet. Med. B. Infect. Dis. Vet. Public Health. 48:695-703.

123. Marois, C., C. Savoye, M. Kobisch, and I. Kempf. 2002. A reverse trancription-PCR assay to detect

viable Mycoplasma synoviae in poultry environmental samples. Vet. Microbiol. 89:17-28.

124. Maslow, J. N., M. E. Mulligan, and R. D. Arbeit. 1993. Molecular epidemiology: application of

contemporary techniques to the typing of microorganisms. Clin. Infect. Dis. 17:153-164.

125. Maule, J. 1998. Pulsed-field gel electrophoresis. Mol. Biotechnol. 9:107-126.


126. McAuliffe, L., R. J. Ellis, R. D. Ayling, and R. A. Nicholas. 2003. Differentiation of Mycoplasma

species by 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis fingerprinting. J. Clin.

Microbiol. 41:4844-4847.

127. McAuliffe, L., B. Kokotovic, R. D. Ayling, and R. A. Nicholas. 2004. Molecular epidemiological

analysis of Mycoplasma bovis isolates from the United Kingdom shows two genetically distinct

clusters. J. Clin. Microbiol. 42:4556-4565.

128. McClelland, M., C. Petersen, and J. Welsh. 1992. Length polymorphisms in tRNA intergenic spacers

detected by using the polymerase chain reaction can distinguish streptococcal strains and species. J.


129. Melin, A. M., A. Allery, A. Perromat, C. Bebear, G. Deleris, and B. de Barbeyrac. 2004. Fourier

transform infrared spectroscopy as a new tool for characterization of mollicutes. J. Microbiol. Methods.

56:73-82.

130. Miyashita, N., A. Saito, S. Kohno, K. Yamaguchi, A. Watanabe, H. Oda, Y. Kazuyama, and T.

Matsushima. 2004. Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae,

Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia. Respir.

Med. 98:542-550.

131. Miyata, M., L. Wang, and T. Fukumura. 1993. Localizing the replication origin region on the

physical map of the Mycoplasma capricolum genome. J. Bacteriol. 175:655-660.

132. Miyata, M., L. Wang, and T. Fukumura. 1991. Physical mapping of the Mycoplasma capricolum

genome. FEMS Microbiol. Lett. 63:329-333.

133. Murchan, S., M. E. Kaufmann, A. Deplano, R. de Ryck, M. Struelens, C. E. Zinn, V. Fussing, S.

Salmenlinna, J. Vuopio-Varkila, N. El Solh, C. Cuny, W. Witte, P. T. Tassios, N. Legakis, W. van

Leeuwen, A. van Belkum, A. Vindel, I. Laconcha, J. Garaizar, S. Haeggman, B. Olsson-Liljequist,

U. Ransjo, G. Coombes, and B. Cookson. 2003. Harmonization of pulsed-field gel electrophoresis

protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single

approach developed by consensus in 10 European laboratories and its application for tracing the spread

of related strains. J. Clin. Microbiol. 41:1574-1585.

134. Nallapareddy, S. R., R. W. Duh, K. V. Singh, and B. E. Murray. 2002. Molecular typing of selected

Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel

electrophoresis. J. Clin. Microbiol. 40:868-876.

135. Ni, H., A. I. Knight, K. A. Cartwright, and J. J. McFadden. 1992. Phylogenetic and epidemiological

analysis of Neisseria meningitidis using DNA probes. Epidemiol. Infect. 109:227-239.

136. Olive, D. M., and P. Bean. 1999. Principles and applications of methods for DNA-based typing of

microbial organisms. J. Clin. Microbiol. 37:1661-1669.


137. Pang, Y., H. Wang, T. Girshick, Z. Xie, and M. I. Khan. 2002. Development and application of a

multiplex polymerase chain reaction for avian respiratory agents. Avian Dis. 46:691-699.

138. Penner, G. A., A. Bush, R. Wise, W. Kim, L. Domier, K. Kasha, A. Laroche, G. Scoles, S. J.

Molnar, and G. Fedak. 1993. Reproducibility of random amplified polymorphic DNA (RAPD)

analysis among laboratories. PCR Methods Appl. 2:341-345.

139. Persson, A., B. Pettersson, G. Bolske, and K. E. Johansson. 1999. Diagnosis of contagious bovine

pleuropneumonia by PCR-laser- induced fluorescence and PCR-restriction endonuclease analysis based

on the 16S rRNA genes of Mycoplasma mycoides subsp. mycoides SC. J. Clin. Microbiol. 37:3815-

3821.

140. Peterson, S. N., T. Lucier, K. Heitzman, E. A. Smith, K. F. Bott, P. C. Hu, and C. A. Hutchison,

3rd. 1995. Genetic map of the Mycoplasma genitalium chromosome. J. Bacteriol. 177:3199-3204.

141. Peterson, S. N., N. Schramm, P. C. Hu, K. F. Bott, and C. A. Hutchison, 3rd. 1991. A random

sequencing approach for placing markers on the physical map of Mycoplasma genitalium. Nucleic

Acids Res. 19:6027-6031.

142. Pettersson, B., T. Leitner, M. Ronaghi, G. Bolske, M. Uhlen, and K. E. Johansson. 1996.

Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA

genes from the two rRNA operons. J Bacteriol. 178:4131-4142.

143. Pillai, S. R., H. L. Mays, Jr., D. H. Ley, P. Luttrell, V. S. Panangala, K. L. Farmer, and S. R.

Roberts. 2003. Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by

sequencing and restriction fragment length polymorphism analysis of the pvpA gene. Avian Dis.

47:640-648.

144. Pilo, P., B. Fleury, M. Marenda, J. Frey, and E. M. Vilei. 2003. Prevalence and distribution of the

insertion element ISMag1 in Mycoplasma agalactiae. Vet. Microbiol. 92:37-48.

145. Pinar, A., N. Bozdemir, T. Kocagoz, and R. Alacam. 2004. Rapid detection of bacterial atypical

pneumonia agents by multiplex PCR. Cent. Eur. J. Public Health. 12:3-5.

146. Pitcher, D., and J. Hilbocus. 1998. Variability in the distribution and composition of insertion

sequence-like elements in strains of Mycoplasma fermentans. FEMS Microbiol. Lett. 160:101-109.

147. Puppe, W., J. A. Weigl, G. Aron, B. Grondahl, H. J. Schmitt, H. G. Niesters, and J. Groen. 2004.

Evaluation of a multiplex reverse transcriptase PCR ELISA for the detection of nine respiratory tract

pathogens. J. Clin. Virol. 30:165-174.

148. Pyle, L. E., and L. R. Finch. 1988. A physical map of the genome of Mycoplasma mycoides

subspecies mycoides Y with some functional loci. Nucleic Acids Res. 16:6027-6039.

149. Pyle, L. E., and L. R. Finch. 1988. Preparation and FIGE separation of infrequent restriction fragments

from Mycoplasma mycoides DNA. Nucleid Acids Res. 16:2263-2268.


150. Radstrom, P., R. Knutsson, P. Wolffs, M. Lovenklev, and C. Lofstrom. 2004. Pre-PCR Processing :

Strategies to generate PCR-compatible samples. Mol. Biotechnol. 26:133-146.

151. Raggam, R. B., E. Leitner, J. Berg, G. Muhlbauer, E. Marth, and H. H. Kessler. 2005. Single-run,

parallel detection of DNA from three pneumonia-producing bacteria by real-time polymerase chain

reaction. J. Mol. Diagn. 7:133-138.

152. Ramisse, V., P. Houssu, E. Hernandez, F. Denoeud, V. Hilaire, O. Lisanti, F. Ramisse, J. D.

Cavallo, and G. Vergnaud. 2004. Variable number of tandem repeats in Salmonella enterica subsp.

enterica for typing purposes. J. Clin. Microbiol. 42:5722-5730.

153. Rawadi, G., and O. Dussurget. 1998. Genotypic methods for diagnosis of mycoplasmal infections in

humans, animals, plants and cell cultures. Biotechnol. Genet. Eng. Rev. 15:51-78.

154. Rawadi, G., B. Lemercier, and D. Roulland-Dussoix. 1995. Application of an arbitrarily-primed

polymerase chain reaction to mycoplasma identification and typing within the Mycoplasma mycoides

cluster. J. Appl. Bacteriol. 78:586-592.

155. Rawadi, G. A. 1998. Characterization of mycoplasmas by RAPD fingerprinting. Methods Mol. Biol.

104:179-187.

156. Razin, S. 1994. DNA probes and PCR in diagnosis of mycoplasma infections. Mol. Cell. Probes.

8:497-511.

157. Razin, S., R. Harasawa, and M. F. Barile. 1983. Cleavage patterns of the mycoplasma chromosome,

obtained by using restriction endonucleases, as indicators of genetic relatedness among strains. Int. J.

Syst. Bacteriol. 33:201-206.



159. Sarkar, S. F., and D. S. Guttman. 2004. Evolution of the core genome of Pseudomonas syringae, a

highly clonal, endemic plant pathogen. Appl. Environ. Microbiol. 70:1999-2012.

160. Sasaki, Y., J. Ishikawa, A. Yamashita, K. Oshima, T. Kenri, K. Furuya, C. Yoshino, A. Horino, T.

Shiba, T. Sasaki, and M. Hattori. 2002. The complete genomic sequence of Mycoplasma penetrans,

an intracellular bacterial pathogen in humans. Nucleic Acids Res. 30:5293-5300.

161. Schaeverbeke, T., M. Clerc, L. Lequen, A. Charron, C. Bebear, B. de Barbeyrac, B. Bannwarth,

and J. Dehais. 1998. Genotypic characterization of seven strains of Mycoplasma fermentans isolated

from synovial fluids of patients with arthritis. J. Clin. Microbiol. 36:1226-1231.

162. Slavec, B., D. Bencina, and P. Dovc. Phylogeny of avian Mycoplasma species based on the dnaK gene

sequence analysis, p. 154 (abstract 262). 15th Congress of the international organization of

mycoplasmology. 2004.

163. Solsona, M., M. Lambert, and F. Poumarat. 1996. Genomic, protein homogeneity and antigenic

variability of Mycoplasma agalactiae. Vet. Microbiol. 50:45-58.


164. Spratt, B. G. 2004. Exploring the concept of clonality in bacteria. Methods Mol. Biol. 266:323-352.

165. Stellrecht, K. A., A. M. Woron, N. G. Mishrik, and R. A. Venezia. 2004. Comparison of multiplex

PCR assay with culture for detection of genital mycoplasmas. J Clin Microbiol. 42:1528-33.

166. Struelens, M. J. 1998. Molecular epidemiologic typing systems of bacterial pathogens: current issues

and perspectives. Mem. Inst. Oswaldo Cruz. 93:581-585.

167. Su, C. J., A. Chavoya, S. F. Dallo, and J. B. Baseman. 1990. Sequence divergency of the cytadhesin

gene of Mycoplasma pneumoniae. Infect. Immun. 58:2669-2674.

168. Supply, P., S. Lesjean, E. Savine, K. Kremer, D. van Soolingen, and C. Locht. 2001. Automated

high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on

mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39:3563-3571.

169. Swaminathan, B., T. J. Barrett, S. B. Hunter, and R. V. Tauxe. 2001. PulseNet: the molecular

subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Dis.

7:382-389.

170. Takahashi-Omoe, H., K. Omoe, S. Matsushita, H. Kobayashi, and K. Yamamoto. 2004.

Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of

Mycoplasma pulmonis in clinical isolates. Comp. Immunol. Microbiol. Infect. Dis. 27:117-128.

171. Tasker, S., C. R. Helps, M. J. Day, T. J. Gruffydd-Jones, and D. A. Harbour. 2003. Use of real-

time PCR to detect and quantify Mycoplasma haemofelis and "Candidatus Mycoplasma

haemominutum" DNA. J. Clin. Microbiol. 41:439-441.

172. Taylor, T. K., J. B. Bashiruddin, and A. R. Gould. 1992. Relationships between members of the

Mycoplasma mycoides cluster as shown by DNA probes and sequence analysis. Int. J. Syst. Bacteriol.

42:593-601.

173. Templeton, K. E., S. A. Scheltinga, A. W. Graffelman, J. M. Van Schie, J. W. Crielaard, P.

Sillekens, P. J. Van Den Broek, H. Goossens, M. F. Beersma, and E. C. Claas. 2003. Comparison

and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional

PCR, and serology for diagnosis of Mycoplasma pneumoniae. J. Clin. Microbiol. 41:4366-4371.

174. Tenover, F. C., R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hebert,

B. Hill, R. Hollis, and et al. 1994. Comparison of traditional and molecular methods of typing isolates

of Staphylococcus aureus. J. Clin. Microbiol. 32:407-415.

175. Tenover, F. C., R. D. Arbeit, and R. V. Goering. 1997. How to select and interpret molecular strain

typing methods for epidemiological studies of bacterial infections: a review for healthcare

epidemiologists. Molecular Typing Working Group of the Society for Healthcare Epidemiology of

America. Infect. Control. Hosp. Epidemiol. 18:426-439.


176. Theiss, P. M., M. F. Kim, and K. S. Wise. 1993. Differential protein expression and surface

presentation generate high-frequency antigenic variation in Mycoplasma fermentans. Infect. Immun.

61:5123-5128.

177. Thomas, A., A. Linden, J. Mainil, D. F. Bischof, J. Frey, and E. M. Vilei. 2005. Mycoplasma bovis

shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides

SC: Evolutionary and developmental aspects. FEMS Microbiol. Lett. 245:249-255.

178. Tigges, E., and F. C. Minion. 1994. Physical map of Mycoplasma gallisepticum. J. Bacteriol.

176:4157-4159.

179. Tjhie, J. H., F. J. van Kuppeveld, R. Roosendaal, W. J. Melchers, R. Gordijn, D. M. MacLaren, J.

M. Walboomers, C. J. Meijer, and A. J. van den Brule. 1994. Direct PCR enables detection of

Mycoplasma pneumoniae in patients with respiratory tract infections. J. Clin. Microbiol. 32:11-16.

180. Tola, S., G. Idini, D. Manunta, I. Casciano, A. M. Rocchigiani, A. Angioi, and G. Leori. 1996.

Comparison of Mycoplasma agalactiae isolates by pulsed field gel electrophoresis, SDS-PAGE and

immunoblotting. FEMS Microbiol. Lett. 143:259-265.

181. Tola, S., G. Idini, A. M. Rocchigiani, D. Manunta, P. P. Angioi, S. Rocca, M. Cocco, and G. Leori.

1999. Comparison of restriction pattern polymorphism of Mycoplasma agalactiae and Mycoplasma

bovis by pulsed field gel electrophoresis. Zentralbl. Veterinarmed. B. 46:199-206.

182. Tola, S., G. Idini, A. M. Rocchigiani, S. Rocca, D. Manunta, and G. Leori. 2001. A physical map of

the Mycoplasma agalactiae strain PG2 genome. Vet. Microbiol. 80:121-130.

183. Tompkins, L. S., N. Troup, A. Labigne-Roussel, and M. L. Cohen. 1986. Cloned, random

chromosomal sequences as probes to identify Salmonella species. J. Infect. Dis. 154:156-162.

184. Tu, A. H., B. Clapper, T. R. Schoeb, A. Elgavish, J. Zhang, L. Liu, H. Yu, and K. Dybvig. 2005.

Association of a major protein antigen of Mycoplasma arthritidis with virulence. Infect. Immun.

73:245-249.

185. Tully, J. G., and S. Razin. 1996. Molecular and diagnostic procedures in mycoplasmology. Section A:

Diagnostic genetic probes, p. 25-75. In J. G. Tully, and S. Razin (ed.), Diagnostic Procedures, vol. II.

Academic Press Inc., San Diego.

186. Tyler, K. D., G. Wang, S. D. Tyler, and W. M. Johnson. 1997. Factors affecting reliability and

reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. J. Clin.


187. Ursi, D., M. Ieven, H. van Bever, W. Quint, H. G. Niesters, and H. Goossens. 1994. Typing of

Mycoplasma pneumoniae by PCR-mediated DNA fingerprinting. J. Clin. Microbiol. 32:2873-2875.

188. Urwin, R., and M. C. Maiden. 2003. Multi-locus sequence typing: a tool for global epidemiology.

Trends Microbiol. 11:479-487.


189. van Belkum, A. 1999. The role of short sequence repeats in epidemiologic typing. Curr. Opin.


190. van Belkum, A., M. Struelens, A. de Visser, H. Verbrugh, and M. Tibayrenc. 2001. Role of

genomic typing in taxonomy, evolutionary genetics, and microbial epidemiology. Clin. Microbiol. Rev.

14:547-560.

191. van Belkum, A., W. van Leeuwen, M. E. Kaufmann, B. Cookson, F. Forey, J. Etienne, R. Goering,

F. Tenover, C. Steward, F. O'Brien, W. Grubb, P. Tassios, N. Legakis, A. Morvan, N. El Solh, R.

de Ryck, M. Struelens, S. Salmenlinna, J. Vuopio-Varkila, M. Kooistra, A. Talens, W. Witte, and

H. Verbrugh. 1998. Assessment of resolution and intercenter reproducibility of results of genotyping

Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a

multicenter study. J. Clin. Microbiol. 36:1653-1659.

192. van der Zee, A., H. Verbakel, J. C. van Zon, I. Frenay, A. van Belkum, M. Peeters, A. Buiting,

and A. Bergmans. 1999. Molecular genotyping of Staphylococcus aureus strains: comparison of

repetitive element sequence-based PCR with various typing methods and isolation of a novel

epidemicity marker. J. Clin. Microbiol. 37:342-349.

193. van Kuppeveld, F. J., J. T. van der Logt, A. F. Angulo, M. J. van Zoest, W. G. Quint, H. G.

Niesters, J. M. Galama, and W. J. Melchers. 1992. Genus- and species-specific identification of

mycoplasmas by 16S rRNA amplification. Appl. Environ. Microbiol. 58:2606-2615.

194. Vaneechoutte, M. 1996. DNA fingerprinting techniques for microorganisms. A proposal for

classification and nomenclature. Mol. Biotechnol. 6:115-142.

195. Vaneechoutte, M. 1999. A plea for caution with regard to applicability of PCR for direct detection. J.

Clin. Microbiol. 37:3081.

196. Vaneechoutte, M., P. Boerlin, H. V. Tichy, E. Bannerman, B. Jager, and J. Bille. 1998. Comparison

of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for

atypical Listeria isolates. Int. J. Syst. Bacteriol. 48 Pt 1:127-139.

197. Vaneechoutte, M., and J. Van Eldere. 1997. The possibilities and limitations of nucleic acid

amplification technology in diagnostic microbiology. J. Med. Microbiol. 46:188-194.

198. Versalovic, J., T. Koeuth, and J. R. Lupski. 1991. Distribution of repetitive DNA sequences in

eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6823-6831.

199. Vilei, E. M., and J. Frey. 2004. Differential clustering of Mycoplasma mycoides subsp. mycoides SC

strains by PCR-REA of the bgl locus. Vet. Microbiol. 100:283-288.

200. Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J.

Peleman, M. Kuiper, and et al. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids

Res. 23:4407-4414.


201. Wang, H., A. A. Fadl, and M. I. Khan. 1997. Multiplex PCR for avian pathogenic mycoplasmas. Mol.

Cell. Probes. 11:211-216.

202. Wang, H., F. Kong, P. Jelfs, G. James, and G. L. Gilbert. 2004. Simultaneous detection and

identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot

hybridization. Appl. Environ. Microbiol. 70:1483-1486.

203. Waring, A. L., T. A. Halse, C. K. Csiza, C. J. Carlyn, K. Arruda Musser, and R. J. Limberger.

2001. Development of a genomics-based PCR assay for detection of Mycoplasma pneumoniae in a

large outbreak in New York State. J. Clin. Microbiol. 39:1385-1390.

204. Weiner, M., and J. Osek. 2003. Comparison of arbitrary primer (AP) PCR and pulsed-field gel

electrophoresis methods for genotyping differentiation of Escherichia coli O157 strains. Bull. Vet. Inst.

Pulaway. 47:363-375.

205. Wellehan, J. F., M. Calsamiglia, D. H. Ley, M. S. Zens, A. Amonsin, and V. Kapur. 2001.

Mycoplasmosis in captive crows and robins from Minnesota. J. Wildl. Dis. 37:547-555.

206. Welsh, J., and M. McClelland. 1991. Genomic fingerprinting using arbitrarily primed PCR and a

matrix of pairwise combinations of primers. Nucleic Acids Res. 19:5275-5279.

207. Welsh, J., and M. McClelland. 1991. Genomic fingerprints produced by PCR with consensus tRNA

gene primers. Nucleic Acids Res. 19:861-866.

208. Welti, M., K. Jaton, M. Altwegg, R. Sahli, A. Wenger, and J. Bille. 2003. Development of a

multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila

and Mycoplasma pneumoniae in respiratory tract secretions. Diagn. Microbiol. Infect. Dis. 45:85-95.

209. Wenzel, R., E. Pirkl, and R. Herrmann. 1992. Construction of an EcoRI restriction map of

Mycoplasma pneumoniae and localization of selected genes. J. Bacteriol. 174:7289-7296.

210. Westberg, J., A. Persson, A. Holmberg, A. Goesmann, J. Lundeberg, K. E. Johansson, B.

Pettersson, and M. Uhlen. 2004. The genome sequence of Mycoplasma mycoides subsp. mycoides SC

type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res.

14:221-227.

211. Westberg, J., A. Persson, B. Pettersson, M. Uhlen, and K. E. Johansson. 2002. ISMmy1, a novel

insertion sequence of Mycoplasma mycoides subsp. mycoides small colony type. FEMS Microbiol. Lett.

208:207-213.

212. Whitley, J. C., A. Muto, and L. R. Finch. 1991. A physical map for Mycoplasma capricolum Cal. kid

with loci for all known tRNA species. Nucleic Acids Res. 19:399-400.

213. Wilton, J. L., A. L. Scarman, M. J. Walker, and S. P. Djordjevic. 1998. Reiterated repeat region

variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae. Microbiology. 144:1931-1943.

214. Woese, C. R., J. Maniloff, and L. B. Zablen. 1980. Phylogenetic analysis of the mycoplasmas. Proc.

Natl. Acad. Sci. USA. 77:494-498.


215. Wolcott, M. J. 1992. Advances in nucleic acid-based detection methods. Clin. Microbiol. Rev. 5:370-

386.

216. Wu, F., and P. Della-Latta. 2002. Molecular typing strategies. Semin. Perinatol. 26:357-366.

217. Yazdankhah, S. P., B. A. Lindstedt, and D. A. Caugant. 2005. Use of variable-number tandem

repeats to examine genetic diversity of Neisseria meningitidis. J. Clin. Microbiol. 43:1699-1705.

218. Yogev, D., D. Halachmi, G. E. Kenny, and S. Razin. 1988. Distinction of species and strains of

mycoplasmas (mollicutes) by genomic DNA fingerprints with an rRNA gene probe. J. Clin. Microbiol.

26:1198-1201.

219. Yogev, D., S. Levisohn, S. H. Kleven, D. Halachmi, and S. Razin. 1988. Ribosomal RNA gene

probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae. Avian Dis.

32:220-231.

220. Yogev, D., D. Menaker, K. Strutzberg, S. Levisohn, H. Kirchhoff, K. H. Hinz, and R.

Rosengarten. 1994. A surface epitope undergoing high-frequency phase variation is shared by

Mycoplasma gallisepticum and Mycoplasma bovis. Infect. Immun. 62:4962-4968.

221. Yoshida, T., S. Maeda, T. Deguchi, and H. Ishiko. 2002. Phylogeny-based rapid identification of

mycoplasmas and ureaplasmas from urethritis patients. J. Clin. Microbiol. 40:105-110.

222. Zariffard, M. R., M. Saifuddin, B. E. Sha, and G. T. Spear. 2002. Detection of bacterial vaginosis-

related organisms by real-time PCR for Lactobacilli, Gardnerella vaginalis and Mycoplasma hominis.

FEMS Immunol. Med. Microbiol. 34:277-281.

223. Zheng, J., and M. A. McIntosh. 1995. Characterization of IS1221 from Mycoplasma hyorhinis:

expression of its putative transposase in Escherichia coli incorporates a ribosomal frameshift

mechanism. Mol. Microbiol. 16:669-685.

65

CHAPTER II

Aims

66 Aims

Since the detection of the first cell wall-less bacteria in 1898, over a hundred fastidious

Mycoplasma spp. have been identified mainly by means of biochemical and serological tests.

These tests have, however, some important limitations for the identification of these bacteria

to the species level. Phenotypic tests are in general not discriminative enough, while

serological cross-reaction is often occurring between related species. Moreover, standardised,

quality-controlled sera for most species are rarely if ever available and laboratories must

depend on in-house prepared sera. To make things worse, for some demanding and slow-

growing Mycoplasma species, isolation is utterly complex and only a few pure colonies are

currently available worldwide. It is therefore impossible for most laboratories to acquire

sufficient expertise in the characterisation of mycoplasmas. As a consequence, their role

during disease was (and probably still is) often overlooked. It is clear that faster, more

reliable, and more attainable tests are needed. With the advent of molecular biology, such

tests have gradually become available. Since most of these tests are species-specific, generally

applicable techniques to handle the wide range of different Mycoplasma spp. are still required.

Therefore, a first general aim of this dissertation was to develop molecular methods for the

identification of mollicutes, especially mycoplasmas.

Mutually, with the rise of molecular identification methods, newly developed techniques for

the demonstration of the diversity of strains emerged. Again owing to the difficulties seen in

the isolation and identification of isolates, their introduction in the field of mycoplasmology

was severely hampered. While some molecular typing methods were optimised for several

species associated with humans or food-producing animals, other important species remained

largely neglected. Also for M. hyopneumoniae, which causes enzootic pneumonia and is

responsible for major economic losses in the pig industry, epidemiological data based on

molecular biology are sparsely available. Moreover, since this organism is extremely difficult

to isolate, typing methods were carried out on only a few isolates. Therefore, a second general

aim of this dissertation was to study the diversity of M. hyopneumoniae isolates by different

molecular typing techniques.

Aims 67

The specific objectives of this dissertation were:

1. to test the applicability of amplified rDNA restriction analysis and tDNA-PCR

for the identification of mollicutes

2. to optimise the isolation and identification of porcine respiratory mycoplasmas

3. to compare both existing and new molecular techniques for the typing of

M. hyopneumoniae isolates

4. to compare the diversity of M. hyopneumoniae strains within a herd and between

different herds

69

CHAPTER III

Experimental

Studies

70 Evaluation of ARDRA for the identification of Mycoplasma species

III.1 EVALUATION OF AMPLIFIED RDNA RESTRICTION

ANALYSIS (ARDRA) FOR THE IDENTIFICATION OF

MYCOPLASMA SPECIES.

Tim Stakenborg1, Jo Vicca2, Patrick Butaye1, Dominiek Maes2, Thierry De Baere3, Rita

Verhelst3, Johan Peeters1, Aart de Kruif1, Freddy Haesebrouck2, and Mario Vaneechoutte3

1 Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium 2 Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke,

Belgium 3 Department of Clinical Chemistry, Microbiology & Immunology, Ghent University

Hospital, De Pintelaan 185, 9000 Ghent, Belgium

Published in: BMC Infectious Diseases (2005) 5(1):46.

Evaluation of ARDRA for the identification of Mycoplasma species 71

Abstract Mycoplasmas are present worldwide in a large number of animal hosts. Due to their small

genome and parasitic lifestyle, Mycoplasma spp. require complex isolation media.

Nevertheless, already over 100 different species have been identified and characterised and

their number increases as more hosts are sampled. We studied the applicability of amplified

rDNA restriction analysis (ARDRA) for the identification of all 116 acknowledged

Mycoplasma species and subspecies. Based upon available 16S rDNA sequences, we

calculated and compared theoretical ARDRA profiles. In silico digestion with the restriction

endonuclease AluI (AG^CT) was found to be most discriminative and generated from 3 to 13

fragments depending on the Mycoplasma species. Although 73 Mycoplasma species could be

differentiated using AluI, other species gave undistinguishable patterns. For these an

additional restriction digestion, typically with BfaI (C^TAG) or HpyF10VI

(GCNNNNN^NNGC), was needed for a final identification. To check the validity of the

theoretically calculated profiles, we performed ARDRA on 60 strains of 27 different species

and subspecies of the genus Mycoplasma. All in vitro obtained restriction profiles were in

accordance with the calculated fragments based on only one 16S rDNA sequence, except for

two isolates of M. columbinum and two isolates of the M. mycoides cluster, for which correct

ARDRA profiles were only obtained if the sequences of both rrn operons were taken into

account. In conclusion, theoretically, restriction digestion of the amplified rDNA was found to

enable differentiation of all described Mycoplasma species and this could be confirmed by

application of ARDRA on a total of 27 species and subspecies.

Introduction Mycoplasmas are phylogenetically related to gram-positive bacteria with low GC-content and

belong to the class of the Mollicutes. They form a unique group of bacteria that lack a

cell-wall and that contain sterols in their cytoplasmatic membrane. They are of great

importance, since several species are pathogenic to animals or humans, whereas species of

other mollicute genera also infect plants and insects (37). In addition, a series of mycoplasmas

cause trouble in the laboratory, because they infect cell cultures. Already over 100 species

have been described, and their number, as well as the number of different hosts is still

increasing.


A correct identification of mycoplasmas, mostly performed after a fastidious initial isolation,

may be achieved by various methods. Original tools to identify mycoplasmas were mainly

based on biochemical and serological differentiation, varying from simple precipitation tests

(15), to ELISA (13, 26), immunofluorescence (3), or Western blot analysis (38). These

techniques are being replaced by faster DNA-based tools (33). Many of these methods are

based on the 16S rDNA sequence for various reasons. First, the 16S rDNA has been

sequenced for all recognised Mycoplasma spp. and is required when describing a new species

(7). Secondly, the 16S rDNA sequences have lower intraspecific variability than most protein

encoding genes, hence their use in the construction of phylogenetic topologies (40). Recently,

denaturing gradient gel electrophoresis of amplified 16S rDNA was shown to be useful to

differentiate most Mycoplasma spp. (27). In another approach, correct identification of related

Mycoplasma spp. was based on differences of the 16S-23S intergenic spacer (ITS) region.

Both size variation (20) as sequence differences (19, 20) of the ITS were successfully used to

differentiate related species. Compared to the 16S rDNA sequence, ITS sequences may vary

more between strains of the same species due to a lower selection pressure (11), although

reports of very highly conserved ITS regions are known as well (8).

Amplified rDNA restriction analysis (ARDRA) has already been used for the identification of

some avian species (16, 18, 22) as well as for pathogenic mycoplasmas in cats (10).

Restriction analysis with PstI of an amplified 16S rDNA fragment was also shown useful to

differentiate M. capricolum subsp. capripneumoniae from the other species belonging to the

mycoides-cluster (6). The potential and power of ARDRA to identify members of the

Mollicutes was already put forward (12), but was never worked out in detail for a large

number of species. In this study, we investigated the value of ARDRA to identify all (to date)

recognised Mycoplasma spp.

Materials and methods

Isolates

A total of 60 strains, belonging to 27 different Mycoplasma species and subspecies, were used

during this study (Table 1). The Mycoplasma spp. belonging to the mycoides-cluster and the

M. hyosynoviae strains, were kindly provided as purified genomic DNA samples by Dr. L.

Manso-Silivan (CIRAD, France) and Dr. B. Kokotovic (DFVF, Denmark), respectively. All

other Mycoplasma spp. were cultivated using F-medium (5), modified Hayflick medium (34),


SP-4-medium (34), SP-4-medium supplemented with L-arginine, HS-medium (17), or Friis’-

medium with ampicillin instead of methicillin (23).

All isolates were previously identified using biochemical tests and growth precipitation tests

with absorbed rabbit antisera (15). Whenever discrepancies existed between the obtained

ARDRA-profiles and the serological results, the 16S rDNA was sequenced for an exact

identification (14).

Table 1: List of strains used in this study.

Mycoplasma species Number of strains

Strain designations

M. agalactiae 2 NCTC 10123 (PG2); 5725 M. arginini 1 884/200 M. bovigenitalium 1 MN120 M. bovirhinis 3 ATCC 27748; O475; CODA 8L M. bovis 4 83/61; 295VD; Widanka309; O422 M. capricolum subsp. capricolum 1 ATCC 27343 (California Kid) M. capricolum subsp. capripneumoniae 1 NCTC 10192 (F38) M. columbinasale 1 397 M. columbinum 4 423VD; 446; 447; 448 M. columborale 1 Pul46 M. dispar 2 ATCC 27140; MdispA M. flocculare 4 ATCC 27399 (Ms42); MP102; MflocF6A; MflocF316 M. gallinarum 3 MgalnA; D63P; MgalnB M. gallisepticum 3 ATCC 19610; A5969; 2000Myc58 M. glycophilum 2 412VD; MglyF1A M. hyopneumoniae 4 ATCC 25934 (J); MhF56C; MhF612D; MhF72C M. hyorhinis 4 MhyorF6A; MhyorF9A; MhyorF7A; MhyorF1A M. hyosynoviae 4 ATCC 25591 (S16); Mp6; Mp96; Mp178 M. lipofaciens 1 R171 M. mycoides subsp. capri 1 Pg3 M. mycoides subsp. mycoides LC 1 YG M. mycoides subsp. mycoides SC 1 Pg1 M. neurolyticum 2 MneuF1A; WVU1853 M. orale 1 ATCC 23714 M. pneumoniae 3 0696A, 1285A, 1284A M. putrefaciens 4 Put85; B387; B731; 7578.95 Mycoplasma sp. bovine group 7 1 Pg50


DNA extraction

DNA of growing cultures was extracted using a phenol-chloroform extraction described

previously (30) or using alkaline lysis. For alkaline lysis, the cultures were centrifuged (2’,

10000 g) and resuspended in 50 µl lysis buffer (0.25% SDS in 0.05 N NaOH). After 5’ at

95°C, 300 µl water was added and the bacterial debris was centrifuged (2’, 10000 g). One µl

of the supernatant was used as template for amplification of the 16S rDNA.

16S PCR amplification

The universal primers pA (5'AGAGTTTGATCCTGGCTCAG) and pH

(5'AAGGAGGTGATCCAGCCGCA) were used to amplify the 16S rRNA genes (14),

yielding an amplification product of approximately 1500 bp. Thirty cycles (20” 94°C; 15”

57°C; and 30’ 72°C) were run on a GeneAmp 9600 Thermal Cycler (Perkin Elmer, USA)

using 3 U recombinant Taq DNA polymerase (Invitrogen, UK), 1x PCR buffer (20 mM Tris-

HCl, 1.5 mM MgCl2, and 50 mM KCl; pH 8.4), 10 pmol of each primer and 1 µl of the

genomic DNA (~30 ng) as template. Reaction volumes were 50 µl.

Restriction digestion

For all 60 strains, 10 µl of the 16S rDNA PCR product was digested with 5 U of restriction

enzyme AluI (Fermentas, Lithuania; sequence: AG^CT) and the associated Y+/Tango

restriction buffer (Fermentas) in a total volume of 20 µl for 2 hours at 37 °C. For a final

identification, the amplified 16S rDNA of some strains were digested in addition with BfaI

(New England Biolabs, USA; sequence: C^TAG) or HpyF10VI (Fermentas; sequence:

GCNNNNN^NNGC). The restriction fragments were separated on a 3% Nusieve 3:1 agar

(Tebu-Bio, France) for 2 hours at 130 V and visualised using a GeneGenius gel

documentation system (Westburg, The Netherlands). A 50-bp ladder was used as a DNA

marker (Fermentas).

Sequences & in silico ARDRA-profiles

ARDRA-profiles were calculated for all Mycoplasma spp. as acknowledged by the

International Committee on Systematics of Prokaryotes (ICPS) to date. The 16S rDNA

sequences were downloaded from Genbank (accession numbers are indicated in Figure 1). A

consensus sequence was constructed and used for species for which more than one sequence

was available. The M. orale 16S rDNA sequence was determined and submitted

[Genbank:AY796060], since the only available sequence contained numerous ambiguities.


For the members of the M. mycoides-cluster - for which differences between rrnA and rrnB

have been published (32) - both sequences were used. For some Mycoplasma spp. only a

partial sequence of the 16S rDNA was available. For these sequences, nucleotides were added

to the 5' and/or 3' ends to generate fragments of expected length. These lengths and the choice

of the nucleotides added were based on a 16S rDNA consensus sequence obtained by

alignment of the complete Mycoplasma 16S rDNA sequences available in Genbank using

Clustal W. The restriction sites and the exact size of the ARDRA fragments were calculated

using Vector NTI Advance V9.0 (Invitrogen) and BioNumerics V3.5 (Applied-Maths,

Belgium).

By way of illustration, a dendrogram, based on ARDRA patterns, was constructed using the

Unweighted Pair Group Method with Arithmetic Means (UPGMA) using 1% tolerance (i.e.

bands that differ about 7 nucleotides or less are considered identical) and taking only

fragments from 80 to 800 nucleotides into account.

Table 2: Number of restriction sites for the members of the M. mycoides-cluster.

Mycoplasma species

Restriction

endonuclease

Mycoplasma sp.

bovine group 7

M. mycoides ssp.

mycoides LC

M. mycoides ssp.

capri

M. mycoides ssp.

mycoides SC

M. capricolum ssp.

capripneumoniae

M. capricolum ssp.

capricolum

BbvI 4 4 4 4 4/2 4 HpyCH4III 3 4 4 3 3 3 HpyF10VI 5 5 5 5 5/4 5 MaeIII 5 5 5 4 5 5 MboII 3/5a 3 3 3 3 3/4 Tsp509I 4 4 4 4/5 4 4 a Two values indicate differences between rrnA and rrnB, based on the Genbank accession numbers indicated in Figure 1.


Figure 1: Theoretical ARDRA patterns after in silico digestion with AluI for all currently

recognised Mycoplasma spp. Patterns are clustered using UPGMA (Bionumerics V3.5) by

way of illustration. The Genbank-accession numbers used are listed together with species

name.


Results For all Mycoplasma spp., the theoretical AluI, BfaI and HpyF10VI restriction patterns were

calculated (Table 3) and are represented in Figure 1-3. For a number of species, ARDRA was

carried out in the laboratory to confirm the in silico obtained results and to check the validity

of the technique for identification. ARDRA profiles obtained with AluI and BfaI are shown in

Figure 4 and Figure 5, respectively. For a further verification of the technique and for the

remaining 9 species that could not be identified with AluI or BfaI alone, ARDRA was also

performed with HpyF10VI (Figure 6, 7).

Figure 2: Calculated ARDRA profiles of Mycoplasma spp. that can be differentiated

using BfaI, but had undistinguishable AluI restriction profiles.


Two of the four M. columbinum strains showed an unpredicted ARDRA pattern after

restriction with AluI. Since the sum of all bands was higher than the length of the 16S

sequence, a difference between the 2 rrn operons was expected. This was verified by

sequence analysis, which revealed an ambiguity at position 997 (i.e. position 1007 in the E.

coli numbering), pointing to the presence of AGCT in one and AGTT in the other operon. As

such, a restriction site for AluI in one operon will lack in the other operon and will lead to a

mixture of ARDRA profiles. Also for the strains of the M. mycoides-cluster the published

sequences of both rrn operons were taken into account (32). By superimposition of the

restriction profiles of both rrnA and rrnB, the correct, expected profiles were obtained.

However, a faint band of approximately 370 nucleotides was observed in the HpyF10VI

restriction profile of M. capricolum subsp. capripneumoniae, indicating a partial restriction at

position 1082 of the rrnA gene (Figure 7). For all other samples, profiles were identical to the

calculated restriction profiles using only one consensus sequence of the Genbank entries.

A few species could not be differentiated with the three suggested enzymes and for these,

other enzymes were selected. M. cricetuli and M. collis, which have 16S rRNA operons that

are 99.8% identical, can be differentiated using Hpy188III. This enzyme cuts the 16S rDNA

of M. collis 7 times, while restriction takes place only 6 times in the 16S rRNA gene of

M. cricetuli. Also the restriction enzyme EarI can be used, since it only restricts the 16S

rRNA gene of M. cricetuli. The very related M. imitans and M. gallisepticum could be

differentiated using MseI or HindII. The restriction enzyme BstUI could be used to

differentiate the otherwise indistinguishable M. haemocanis (2 restriction sites) and

M. haemofelis (3 restriction sites). The determined 16S rDNA sequence of M. orale was

almost identical to the 16S rDNA of M. indiense and specific restriction enzymes, like BsaJI

or EcoHI, were necessary to differentiate these species. In case of the very related members of

the mycoides-cluster, the differentiation is more complicated and a whole series of restrictions

are needed. Based on the occurrence of different restriction sites, it is however theoretically

possible to correctly identify these species as well, using only commercially available

restriction endonucleases (Table 2).


Figure 3: Calculated ARDRA profiles of Mycoplasma spp. that can be differentiated

using HpyF10VI, but had undistinguishable AluI restriction profiles. The restriction

pattern of M. capricolum subsp. capricolum represents the not included members of

the M. mycoides-cluster as well.


Figure 4: ARDRA profiles after restriction with AluI of 18 different Mycoplasma species.

Since all samples of the same species gave identical restriction patterns, the number of strains

tested for each species is indicated in parenthesis. A Generuler 50-bp ladder (Fermentas) was

used as size-marker.

50-b

p G

ener

eule

r

M. g

allis

eptic

um (3

)

M. p

neum

onia

e (3

)

M. g

lyco

philu

m (2

)

M. b

ovir

hini

s (2)

M. c

olum

bora

le (1

)

M. n

euro

lytic

um (2

)

M. f

locc

ular

e (4

)

M. d

ispa

r (2)

M. h

yopn

eum

onia

e (4

)

M. h

yorh

inis

(4)

M. a

rgin

ini (

1)

M. h

yosy

novi

ae (4

)

M. o

rale

(1)

M. c

olum

bina

sale

(1)

M. a

gala

ctia

e (2

)

M. b

ovis

(4)

M. l

ipof

acie

ns (1

)

M. g

allin

arum

(3)

50-b

p G

ener

eule

r

1000 900 800 700

600

500

400

300

250

200

150

100

50


Figure 5: ARDRA profiles after restriction with BfaI of 18 different Mycoplasma species.

Since all samples of the same species gave identical restriction patterns, the number of strains

tested for each species is indicated in parenthesis. A Generuler 50-bp ladder (Fermentas) was

used as size-marker.

50-b

p G

ener

eule

r

M. g

allis

eptic

um (3

)

M. p

neum

onia

e (3

)

M. g

lyco

philu

m (2

)

M. b

ovir

hini

s (2)

M. c

olum

bora

le (1

)

M. n

euro

lytic

um (2

)

M. f

locc

ular

e (4

)

M. d

ispa

r (2)

M. h

yopn

eum

onia

e (4

)

M. h

yorh

inis

(4)

M. a

rgin

ini (

1)

M. h

yosy

novi

ae (4

)

M. o

rale

(1)

M. c

olum

bina

sale

(1)

M. a

gala

ctia

e (2

)

M. b

ovis

(4)

M. l

ipof

acie

ns (1

)

M. g

allin

arum

(3)

50-b

p G

ener

eule

r

1000 900 800 700

600

500

400

300

250

200

150

100

50


Figure 6: ARDRA profiles after restriction with AluI (left) or HpyF10VI (right) of M.

bovigenitalium and of M. columbinum. A Generuler 50-bp ladder (Fermentas) was used as

size-marker. The number of strains tested for each species is indicated in parenthesis.

50-b

p G

ener

eule

r

M. b

ovig

enita

lium

(1)

M. c

olum

binu

m A

(2)

M. c

olum

binu

m B

(2)

50-b

p G

ener

eule

r

M. b

ovig

enita

lium

(1)

M. c

olum

binu

m A

(2)

M. c

olum

binu

m B

(2)

500

400

300

250

200

150

100

50


Figure 7: ARDRA profiles of M. putrefaciens and the M. mycoides cluster after restriction

with AluI (left) and HpyF10VI (right). The expected band sizes for both rrn operons are

indicated in Table 2. An O’RangeRuler 50-bp ladder (Fermentas) was used as size-marker.

The number of strains tested for each species is indicated in parenthesis.

50-b

p O

’Ran

geR

uler

M. p

utre

faci

ens (

4)

M. c

apri

colu

m sp

p. c

apri

colu

m (1

)

M. c

apri

colu

m sp

p. c

apri

pneu

mon

iae

(1)

M. m

ycoi

des s

pp. m

ycoi

des c

apri

(1)

M. m

ycoi

des s

pp. m

ycoi

des L

C (1

)

M. m

ycoi

des s

pp. m

ycoi

des S

C (1

)

M. s

p. b

ovin

e gr

oup

7 (1

)

50-b

p O

’Ran

geR

uler

M. p

utre

faci

ens (

4)

M. c

apri

colu

m sp

p. c

apri

colu

m (1

)

M. c

apri

colu

m sp

p. c

apri

pneu

mon

iae

(1)

M. m

ycoi

des s

pp. m

ycoi

des c

apri

(1)

M. m

ycoi

des s

pp. m

ycoi

des L

C (1

)

M. m

ycoi

des s

pp. m

ycoi

des S

C (1

)

M. s

p. b

ovin

e gr

oup

7 (1

)

1000

700

500

400

300

250

200

150

100

50

Tab

le 3

: Ove

rvie

w o

f th

e re

stric

tion

frag

men

ts (

and

corr

espo

ndin

g re

stric

tion

site

s) a

fter

AR

DR

A w

ith A

luI,

BfaI

and

Hpy

F10V

I fo

r al

l

curr

ent 1

16 M

ycop

lasm

a sp

ecie

s an

d su

bspe

cies

1 . The

rest

rictio

n en

zym

es n

eede

d to

obt

ain

a co

rrec

t ide

ntifi

catio

n ar

e m

arke

d in

bol

d2 . The

frag

men

ts a

re li

sted

acc

ordi

ng to

thei

r siz

e.

R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. a

dler

i 35

2 (3

70-7

21),

291

(104

4-13

34),

232

(1-2

32),

147

(841

-987

), 13

7 (2

33-3

69),

112

(139

4-15

05),

95 (

722-

816)

, 59

(133

5-13

93),

56 (

988-

1043

), 24

(817

-840

)

681

(637

-131

7), 4

03 (2

34-6

36),

233

(1-2

33),

188

(131

8-15

05)

489

(230

-718

), 23

7 (8

44-1

080)

, 187

(12

13-1

399)

, 164

(66

-229

), 13

2 (1

081-

1212

), 10

6 (1

400-

1505

), 81

(763

-843

), 56

(1-5

6), 4

4 (7

19-7

62),

9 (5

7-65

)

M. a

gala

ctia

e 48

9 (2

34-7

22),

291

(104

5-13

35),

233

(1-2

33),

147

(842

-988

), 11

9 (7

23-8

41),

112

(139

5-15

06),

59 (1

336-

1394

), 56

(989

-104

4),

681

(638

-131

8),

403

(235

-637

), 20

7 (1

-207

), 18

8 (1

319-

1506

), 27

(2

08-2

34)

489

(231

-719

), 31

9 (1

082-

1400

), 23

7 (8

45-1

081)

, 165

(66

-230

), 10

6 (1

401-

1506

), 81

(764

-844

), 56

(1-5

6), 3

5 (7

29-7

63),

9 (5

7-65

), 9

(720

-72

8)

M. a

gass

izii

72

0 (1

-720

), 29

1 (1

046-

1336

), 14

7 (8

43-9

89),

122

(721

-842

), 95

(1

421-

1515

), 84

(133

7-14

20),

56(9

90-1

045)

68

2 (6

38-1

319)

, 564

(74-

637)

, 196

(132

0-15

15),

65 (1

-65)

, 8 (6

6-73

) 52

5 (1

93-7

17),

237

(846

-108

2), 1

86 (

1216

-140

1), 1

36 (

57-1

92),

133

(108

3-12

15),

114

(140

2-15

15),

78 (7

62-8

39),

56 (1

-56)

, 44

(718

-761

), 6;

840

-845

)

M. a

lkal

esce

ns

293

(104

2-13

34),

255

(465

-719

), 20

1 (1

-201

), 16

9 (2

02-3

70),

147

(839

-985

), 11

9 (7

20-8

38),

96 (

1418

-151

3),

94 (

371-

464)

, 59

(13

35-

1393

), 56

(986

-104

1), 2

4 (1

394-

1417

)

487

(637

-112

3),

429

(208

-636

), 19

6 (1

318-

1513

), 19

4 (1

124-

1317

), 13

2 (7

6-20

7), 7

5 (1

-75)

51

8 (1

99-7

16),

435

(107

9-15

13),

237

(842

-107

8),

101

(57-

157)

, 81

(7

61-8

41),

56 (1

-56)

, 44

(717

-760

), 41

(158

-198

)

M. a

lliga

tori

s 43

5 (1

68-6

02),

277

(104

1-13

17),

203

(838

-104

0),

167

(1-1

67),

121

(137

7-14

97),

118

(603

-720

), 95

(72

1-81

5), 5

9 (1

318-

1376

), 22

(81

6-83

7)

501

(135

-635

), 47

1 (6

36-1

106)

, 19

7 (1

301-

1497

), 19

4 (1

107-

1300

), 13

4 (1

-134

) 56

8 (1

50-7

17),

305

(107

8-13

82),

237

(841

-107

7), 1

15 (1

383-

1497

), 79

(7

62-8

40),

56 (1

-56)

, 53

(57-

109)

, 40

(110

-149

), 35

(727

-761

), 9

(718

-72

6)

M. a

lvi

612

(235

-846

), 19

2 (1

051-

1242

), 14

8 (8

47-9

94),

146

(1-1

46),

143

(124

3-13

85),

122

(138

6-15

07),

88 (1

47-2

34),

56 (9

95-1

050)

39

9 (2

36-6

34),

186

(786

-971

), 14

6 (6

40-7

85),

145

(972

-111

6),

126

(118

4-13

09),

123

(131

0-14

32),

104

(132

-235

), 76

(1-

76),

75 (

1433

-15

07),

67 (1

117-

1183

), 55

(77-

131)

, 5 (6

35-6

39)

669

(181

-849

), 30

2 (1

206-

1507

), 23

8 (8

50-1

087)

, 15

2 (1

-152

), 11

8 (1

088-

1205

), 28

(153

-180

)

M. a

natis

27

7 (1

043-

1319

), 23

2 (1

-232

), 14

7 (8

40-9

86),

121

(137

9-14

99),

95

(721

-815

), 59

(132

0-13

78),

56 (9

87-1

042)

, 24

(816

-839

) 40

2 (2

34-6

35),

206

(1-2

06),

197

(130

3-14

99),

27 (2

07-2

33)

661

(57-

717)

, 318

(762

-107

9), 3

05 (

1080

-138

4), 1

15 (1

385-

1499

), 56

(1

-56)

, 35

(727

-761

), 9

(718

-726

)

M. a

nser

is

293

(103

9-13

31),

255

(463

-717

), 19

9 (1

-199

), 16

9 (2

00-3

68),

147

(836

-982

), 12

0 (1

391-

1510

), 11

8 (7

18-8

35),

94 (

369-

462)

, 59

(133

2-13

90),

56 (9

83-1

038)

429

(206

-634

), 19

6 (1

315-

1510

), 19

4 (1

121-

1314

), 13

1 (7

5-20

5), 7

4 (1

-74)

65

8 (5

7-71

4), 4

35 (1

076-

1510

), 23

7 (8

39-1

075)

, 80

(759

-838

), 56

(1-

56),

44 (7

15-7

58)

M. a

rgin

ini

350

(373

-722

), 29

3 (1

045-

1337

), 20

3 (1

-203

), 20

3 (8

42-1

044)

, 16

9 (2

04-3

72),

119

(723

-841

), 95

(142

1-15

15),

59 (1

338-

1396

), 24

(139

7-14

20)

487

(640

-112

6),

430

(210

-639

), 19

5 (1

321-

1515

), 19

4 (1

127-

1320

), 13

2 (7

8-20

9), 7

7 (1

-77)

51

9 (2

01-7

19),

434

(108

2-15

15),

237

(845

-108

1),

102

(58-

159)

, 81

(7

64-8

44),

57 (1

-57)

, 44

(720

-763

), 41

(160

-200

)

M. a

rthr

itidi

s 29

3 (1

042-

1334

), 25

5 (4

65-7

19),

201

(1-2

01),

137

(234

-370

), 12

0 (1

394-

1513

), 11

9 (7

20-8

38),

105

(839

-943

), 94

(37

1-46

4), 5

9 (1

335-

1393

), 56

(986

-104

1), 4

2 (9

44-9

85),

32 (2

02-2

33)

487

(637

-112

3),

391

(208

-598

), 20

7 (1

-207

), 19

6 (1

318-

1513

), 19

4 (1

124-

1317

), 38

(599

-636

) 55

8 (1

59-7

16),

435

(107

9-15

13),

237

(842

-107

8),

93 (

66-1

58),

81

(761

-841

), 56

(1-5

6), 4

4 (7

17-7

60),

9 (5

7-65

)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. a

uris

37

0 (1

-370

), 29

3 (1

042-

1334

), 25

5 (4

65-7

19),

147

(839

-985

), 11

9 (7

20-8

38),

96 (

1418

-151

3),

94 (

371-

464)

, 59

(13

35-1

393)

, 56

(98

6-10

41),

24 (1

394-

1417

)

561

(76-

636)

, 487

(63

7-11

23),

196

(131

8-15

13),

194

(112

4-13

17),

75

(1-7

5)

518

(199

-716

), 43

5 (1

079-

1513

), 23

7 (8

42-1

078)

, 10

1 (5

7-15

7),

81

(761

-841

), 56

(1-5

6), 4

4 (7

17-7

60),

41 (1

58-1

98)

M. b

ovig

enita

lium

48

9 (2

35-7

23),

291

(104

6-13

36),

234

(1-2

34),

147

(843

-989

), 11

2 (1

396-

1507

), 95

(72

4-81

8), 5

9 (1

337-

1395

), 56

(99

0-10

45),

24 (

819-

842)

487

(639

-112

5),

403

(236

-638

), 23

5 (1

-235

), 19

4 (1

126-

1319

), 18

8 (1

320-

1507

) 48

9 (2

32-7

20),

237

(846

-108

2), 1

87 (

1215

-140

1), 1

57 (

57-2

13),

132

(108

3-12

14),

106

(140

2-15

07),

81 (7

65-8

45),

56 (1

-56)

, 44

(721

-764

), 9

(214

-222

), 9

(223

-231

)

M. b

ovir

hini

s 48

8 (2

35-7

22),

276

(104

6-13

21),

202

(1-2

02),

147

(843

-989

), 12

1 (1

381-

1501

), 95

(72

3-81

7), 5

9 (1

322-

1380

), 56

(99

0-10

45),

32 (

203-

234)

, 25

(818

-842

)

638

(667

-130

4),

235

(1-2

35),

223

(236

-458

), 19

7 (1

305-

1501

), 17

9 (4

59-6

37),

29 (6

38-6

66)

663

(57-

719)

, 304

(10

83-1

386)

, 237

(84

6-10

82),

115

(138

7-15

01),

82

(764

-845

), 56

(1-5

6), 3

5 (7

29-7

63),

9 (7

20-7

28)

M. b

ovis

48

9 (2

34-7

22),

291

(104

5-13

35),

233

(1-2

33),

147

(842

-988

), 11

9 (7

23-8

41),

112

(139

5-15

06),

59 (1

336-

1394

), 56

(989

-104

4)

681

(638

-131

8), 4

03 (2

35-6

37),

234

(1-2

34),

188

(131

9-15

06)

489

(231

-719

), 31

9 (1

082-

1400

), 23

7 (8

45-1

081)

, 165

(66

-230

), 10

6 (1

401-

1506

), 81

(764

-844

), 56

(1-5

6), 3

5 (7

29-7

63),

9 (5

7-65

), 9

(720

-72

8)

M. b

ovoc

uli

233

(382

-614

), 19

0 (1

155-

1344

), 17

9 (1

345-

1523

), 17

7 (2

05-3

81),

146

(852

-997

), 12

0 (7

32-8

51),

117

(615

-731

), 10

1 (1

054-

1154

), 84

(73

-15

6), 7

2 (1

-72)

, 56

(998

-105

3), 4

8 (1

57-2

04)

457

(220

-676

), 29

0 (8

44-1

133)

, 21

9 (1

-219

), 19

6 (1

328-

1523

), 19

4 (1

134-

1327

), 16

7(67

7-84

3)

728

(1-7

28),

318

(773

-109

0), 3

01 (

1223

-152

3), 1

32 (

1091

-122

2), 4

4 (7

29-7

72)

M. b

ucca

le

293

(104

1-13

33),

257

(463

-719

), 23

1 (1

-231

), 20

3 (8

38-1

040)

, 13

7 (2

32-3

68),

120

(139

3-15

12),

118

(720

-837

), 94

(36

9-46

2), 5

9 (1

334-

1392

)

634

(1-6

34),

488

(635

-112

2), 1

96 (1

317-

1512

), 19

4(11

23-1

316)

55

8 (1

59-7

16),

435

(107

8-15

12),

237

(841

-107

7),

93 (

66-1

58),

80

(761

-840

), 56

(1-5

6), 4

4 (7

17-7

60),

9 (5

7-65

)

M. b

uteo

nis

370

(233

-602

), 27

7 (1

041-

1317

), 23

2 (1

-232

), 20

3 (8

38-1

040)

, 12

1 (1

377-

1497

), 11

8 (6

03-7

20),

95 (

721-

815)

, 59

(131

8-13

76),

22 (

816-

837)

442

(665

-110

6),

402

(234

-635

), 20

6 (1

-206

), 19

7 (1

301-

1497

), 19

4 (1

107-

1300

), 29

(636

-664

), 27

(207

-233

) 66

1 (5

7-71

7), 4

20 (

1078

-149

7), 2

37 (

841-

1077

), 79

(76

2-84

0), 5

6 (1

-56

), 35

(727

-761

), 9

(718

-726

)

M. c

alifo

rnic

um

488

(234

-721

), 29

1 (1

044-

1334

), 23

3 (1

-233

), 14

7 (8

41-9

87),

112

(139

4-15

05),

95 (

722-

816)

, 59

(133

5-13

93),

56 (

988-

1043

), 24

(81

7-84

0)

681

(637

-131

7), 4

02 (2

35-6

36),

234

(1-2

34),

188(

1318

-150

5)

488

(231

-718

), 23

7 (8

44-1

080)

, 187

(12

13-1

399)

, 142

(57

-198

), 13

2 (1

081-

1212

), 10

6 (1

400-

1505

), 81

(763

-843

), 56

(1-5

6), 4

4 (7

19-7

62),

14 (1

99-2

12),

9 (2

13-2

21),

9 (2

22-2

30)

M. c

anad

ense

29

3 (1

043-

1335

), 25

5 (4

66-7

20),

203

(840

-104

2),

202

(1-2

02),

169

(203

-371

), 11

9 (7

21-8

39),

96 (

1419

-151

4),

94 (

372-

465)

, 59

(13

36-

1394

), 24

(139

5-14

18)

561

(77-

637)

, 487

(63

8-11

24),

196

(131

9-15

14),

194

(112

5-13

18),

76

(1-7

6)

518

(200

-717

), 43

5 (1

080-

1514

), 23

7 (8

43-1

079)

, 10

2 (5

7-15

8),

81

(762

-842

), 56

(1-5

6), 4

4 (7

18-7

61),

41 (1

59-1

99)

M. c

anis

48

8 (2

36-7

23),

276

(104

6-13

21),

203

(1-2

03),

147

(843

-989

), 12

2 (1

381-

1502

), 95

(72

4-81

8), 5

9 (1

322-

1380

), 56

(99

0-10

45),

32 (

204-

235)

, 24

(819

-842

)

443

(668

-111

0),

402

(237

-638

), 23

6 (1

-236

), 19

8 (1

305-

1502

), 19

4 (1

111-

1304

), 29

(639

-667

) 52

0 (2

01-7

20),

318

(765

-108

2), 3

04 (

1083

-138

6), 1

44 (

57-2

00),

116

(138

7-15

02),

56 (1

-56)

, 35

(730

-764

), 9

(721

-729

)

M. c

apri

colu

m ss

p.

capr

icol

um

236

(605

-840

), 23

4 (1

-234

), 18

6 (2

35-4

20),

184

(421

-604

), 15

7 (9

88-

1144

), 14

7 (8

41-9

87),

105

(114

5-12

49),

99 (

1417

-151

5),

85 (

1250

-13

34),

82(1

335-

1416

)

378

(260

-637

), 35

2 (7

84-1

135)

, 23

5 (1

-235

), 17

2 (1

146-

1317

), 14

6 (6

38-7

83),

134

(131

8-14

51),

64 (1

452-

1515

), 24

(236

-259

), 10

(113

6-11

45)

717

(1-7

17),

303

(121

3-15

15),

237

(844

-108

0), 1

32 (

1081

-121

2), 8

2 (7

62-8

43),

44 (7

18-7

61)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. c

apri

colu

m ss

p.

capr

ipne

umon

iae

262

(98

8-12

49)b , 2

36 (

605-

840)

, 23

4 (1

-234

) , 1

86 (

235-

420)

, 184

(4

21-6

04),

157

(988

-114

4)a ,

147

(841

-987

), 10

5 (1

145-

1249

)a , 99

(1

417-

1515

), 85

(125

0-13

34),

82 (1

335-

1416

)

378

(260

-637

), 35

2 (7

84-1

135)

, 235

(1-

235)

, 182

(11

36-1

317)

b , 172

(1

146-

1317

)a , 14

6 (6

38-7

83),

134

(131

8-14

51),

64 (

1452

-151

5),

24

(236

-259

), 10

(113

6-11

45)a

717

(1-7

17),

342

(871

-121

2)b , 3

03 (1

213-

1515

), 23

7 (8

44-1

080)

a , 132

(1

081-

1212

)a , 109

(762

-870

)b , 82

(762

-843

)a , 44

(718

-761

)

M. c

avia

e

489

(233

-721

), 29

1 (1

044-

1334

), 23

2 (1

-232

), 14

7 (8

41-9

87),

112

(139

4-15

05),

95 (

722-

816)

, 59

(133

5-13

93),

56 (

988-

1043

), 24

(817

-84

0)

681

(637

-131

7), 4

03 (2

34-6

36),

233

(1-2

33),

188

(131

8-15

05)

653

(66-

718)

, 23

7 (8

44-1

080)

, 18

7 (1

213-

1399

), 13

2 (1

081-

1212

), 10

6 (1

400-

1505

), 81

(763

-843

), 56

(1-5

6), 4

4 (7

19-7

62),

9 (5

7-65

)

M. c

avip

hary

ngis

32

9 (2

74-6

02),

273

(1-2

73),

240

(603

-842

), 18

9 (1

131-

1319

), 18

4 (1

320-

1503

), 14

7 (8

43-9

89),

85 (1

046-

1130

), 56

(990

-104

5)

635

(1-6

35),

479

(824

-130

2),

146

(636

-781

), 12

6 (1

303-

1428

), 75

(1

429-

1503

), 42

(782

-823

) 84

5 (1

-845

), 42

1 (1

083-

1503

), 23

7 (8

46-1

082)

M. c

itelli

44

6 (3

72-8

17),

277

(104

3-13

19),

234

(1-2

34),

147

(840

-986

), 12

1 (1

379-

1499

), 11

8 (2

54-3

71),

59 (1

320-

1378

), 56

(987

-104

2), 2

2 (8

18-

839)

, 19

(235

-253

)

484

(638

-112

1),

402

(236

-637

), 23

5 (1

-235

), 19

7 (1

303-

1499

), 18

1 (1

122-

1302

) 70

7 (5

7-76

3), 3

05 (1

080-

1384

), 23

7 (8

43-1

079)

, 115

(138

5-14

99),

79

(764

-842

), 56

(1-5

6)

M. c

loac

ale

29

3 (1

040-

1332

), 25

5 (4

64-7

18),

200

(1-2

00),

147

(837

-983

), 13

7 (2

33-3

69),

120

(139

2-15

11),

118

(719

-836

), 94

(37

0-46

3), 5

9 (1

333-

1391

), 56

(984

-103

9), 3

2 (2

01-2

32)

486

(636

-112

1),

364

(234

-597

), 23

3 (1

-233

), 19

6 (1

316-

1511

), 19

4 (1

122-

1315

), 38

(598

-635

) 51

8 (1

98-7

15),

435

(107

7-15

11),

237

(840

-107

6),

141

(57-

197)

, 74

(7

60-8

33),

56 (1

-56)

, 44

(716

-759

), 6

(834

-839

)

M. c

ollis

29

1 (1

058-

1348

), 23

3 (3

85-6

17),

203

(855

-105

7),

202

(1-2

02),

182

(203

-384

), 12

0 (7

35-8

54),

117

(618

-734

), 96

(14

33-1

528)

, 84

(134

9-14

32)

679

(1-6

79),

458

(680

-113

7), 1

97 (1

332-

1528

), 19

4 (1

138-

1331

),

731

(1-7

31),

434

(109

5-15

28),

237

(858

-109

4),

82 (

776-

857)

, 44

(7

32-7

75)

M. c

olum

bina

sale

48

9 (2

35-7

23),

291

(104

6-13

36),

203

(843

-104

5),

120

(1-1

20),

114

(121

-234

), 11

2 (1

396-

1507

), 95

(724

-818

), 59

(13

37-1

395)

, 24

(819

-84

2)

487

(639

-112

5),

403

(236

-638

), 23

5 (1

-235

), 19

4 (1

126-

1319

), 18

8 (1

320-

1507

) 48

9 (2

32-7

20),

319

(108

3-14

01),

237

(846

-108

2), 1

75 (

57-2

31),

106

(140

2-15

07),

81 (7

65-8

45),

56 (1

-56)

, 44

(721

-764

)

M. c

olum

binu

m

490

(233

-722

), 29

1 (1

045-

1335

), 23

2 (1

-232

), 14

7 (8

42-9

88),

112

(139

5-15

06),

95 (

723-

817)

, 59

(133

6-13

94),

56 (

989-

1044

), 24

(818

-84

1)

681

(638

-131

8), 4

04 (2

34-6

37),

233

(1-2

33),

188

(131

9-15

06)

490

(230

-719

), 31

9 (1

082-

1400

), 23

7 (8

45-1

081)

, 155

(57

-211

), 10

6 (1

401-

1506

), 81

(76

4-84

4), 5

6 (1

-56)

, 44

(720

-763

), 9

(212

-220

), 9

(221

-229

)

M. c

olum

bora

le

446

(371

-816

), 27

7 (1

042-

1318

), 23

3 (1

-233

), 14

7 (8

39-9

85),

137

(234

-370

), 12

1 (1

378-

1498

), 59

(131

9-13

77),

56 (9

86-1

041)

, 22

(817

-83

8)

665

(637

-130

1),

402

(235

-636

), 19

7 (1

302-

1498

), 11

8 (1

-118

), 89

(1

19-2

07),

27 (2

08-2

34)

706

(57-

762)

, 305

(107

9-13

83),

237

(842

-107

8), 1

15 (1

384-

1498

), 79

(7

63-8

41),

56 (1

-56)

M. c

onju

nctiv

ae

292

(105

6-13

47),

233

(383

-615

), 17

9 (1

348-

1526

), 16

9 (2

14-3

82),

156

(1-1

56),

146

(854

-999

), 12

1 (7

33-8

53),

117

(616

-732

), 57

(15

7-21

3), 5

6 (1

000-

1055

)

653

(678

-133

0), 4

57 (2

21-6

77),

220

(1-2

20),

196

(133

1-15

26)

519

(211

-729

), 31

9 (7

74-1

092)

, 30

1 (1

226-

1526

), 21

0 (1

-210

), 13

3 (1

093-

1225

), 44

(730

-773

)

M. c

orag

ypsi

48

8 (2

32-7

19),

277

(104

0-13

16),

231

(1-2

31),

225

(815

-103

9),

121

(137

6-14

96),

95 (7

20-8

14),

59 (1

317-

1375

) 44

2 (6

64-1

105)

, 40

2 (2

33-6

34),

232

(1-2

32),

197

(130

0-14

96),

194

(110

6-12

99),

29 (6

35-6

63)

660

(57-

716)

, 305

(107

7-13

81),

136

(840

-975

), 11

5 (1

382-

1496

), 10

1 (9

76-1

076)

, 79

(761

-839

), 56

(1-5

6), 3

5 (7

26-7

60),

9 (7

17-7

25)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. c

otte

wii

23

7 (6

05-8

41),

234

(1-2

34),

186

(235

-420

), 18

4 (4

21-6

04),

157

(989

-11

45),

147

(842

-988

), 10

5 (1

146-

1250

), 99

(14

18-1

516)

, 85

(12

51-

1335

), 82

(133

6-14

17)

378

(260

-637

), 32

4 (8

13-1

136)

, 23

5 (1

-235

), 19

8 (1

319-

1516

), 17

2 (1

147-

1318

), 14

6 (6

38-7

83),

29 (

784-

812)

, 24

(236

-259

), 10

(11

37-

1146

)

392

(1-3

92),

316

(402

-717

), 23

7 (8

45-1

081)

, 21

8 (1

214-

1431

), 13

2 (1

082-

1213

), 85

(14

32-1

516)

, 83

(76

2-84

4),

44 (

718-

761)

, 9

(393

-40

1)

M. c

rice

tuli

29

0 (1

058-

1347

), 23

3 (3

85-6

17),

203

(855

-105

7),

202

(1-2

02),

182

(203

-384

), 12

0 (7

35-8

54),

117

(618

-734

), 97

(14

32-1

528)

, 84

(134

8-14

31)

679

(1-6

79),

458

(680

-113

7), 1

98 (1

331-

1528

), 19

3 (1

138-

1330

) 73

1 (1

-731

), 43

4 (1

095-

1528

), 23

7 (8

58-1

094)

, 82

(77

6-85

7),

44

(732

-775

)

M. c

roco

dyli

60

2 (1

-602

), 27

7 (1

040-

1316

), 14

7 (8

37-9

83),

121

(137

6-14

96),

95

(720

-814

), 85

(63

5-71

9),

59 (

1317

-137

5),

56 (

984-

1039

), 32

(60

3-63

4), 2

2 (8

15-8

36)

635

(1-6

35),

470

(636

-110

5), 1

97 (1

300-

1496

), 19

4 (1

106-

1299

) 60

7 (1

10-7

16),

305

(107

7-13

81),

237

(840

-107

6), 1

15 (

1382

-149

6),

79 (7

61-8

39),

56 (1

-56)

, 53

(57-

109)

, 35

(726

-760

), 9

(717

-725

)

M. c

ynos

48

8 (2

36-7

23),

276

(104

6-13

21),

195

(1-1

95),

147

(843

-989

), 12

1 (1

381-

1501

), 95

(72

4-81

8), 5

9 (1

322-

1380

), 56

(99

0-10

45),

32 (2

04-

235)

, 24

(819

-842

), 8

(196

-203

)

637

(668

-130

4),

402

(237

-638

), 23

6 (1

-236

), 19

7 (1

305-

1501

), 29

(639

-667

) 66

4 (5

7-72

0), 3

04 (1

083-

1386

), 23

7 (8

46-1

082)

, 115

(138

7-15

01),

81

(765

-845

), 56

(1-5

6), 3

5 (7

30-7

64),

9 (7

21-7

29)

M. d

ispa

r 23

3 (3

83-6

15),

206

(105

5-12

60),

181

(33-

213)

, 179

(134

6-15

24),

169

(214

-382

), 14

6 (8

53-9

98),

120

(733

-852

), 11

7 (6

16-7

32),

85 (

1261

-13

45),

56 (9

99-1

054)

, 32

(1-3

2)

378

(78-

455)

, 290

(84

5-11

34),

206

(472

-677

), 19

6 (1

329-

1524

), 19

4 (1

135-

1328

), 16

7 (6

78-8

44),

40 (

30-6

9), 2

9 (1

-29)

, 16

(456

-471

), 8

(70-

77),

519

(211

-729

), 31

8 (7

74-1

091)

, 30

1 (1

224-

1524

), 21

0 (1

-210

), 13

2 (1

092-

1223

), 44

(730

-773

)

M. e

dwar

dii

402

(235

-636

), 27

6 (1

044-

1319

), 19

4 (1

-194

), 14

7 (8

41-9

87),

121

(137

9-14

99),

95 (

723-

817)

, 86

(637

-722

), 59

(13

20-1

378)

, 56

(988

-10

43),

40 (1

95-2

34),

23 (8

18-8

40)

442

(667

-110

8),

402

(236

-637

), 23

5 (1

-235

), 19

7 (1

303-

1499

), 19

4 (1

109-

1302

), 29

(638

-666

) 66

3 (5

7-71

9), 3

04 (1

081-

1384

), 23

7 (8

44-1

080)

, 115

(138

5-14

99),

80

(764

-843

), 56

(1-5

6), 3

5 (7

29-7

63),

9 (7

20-7

28)

M. e

leph

antis

34

9 (3

68-7

16),

291

(103

9-13

29),

203

(836

-103

8), 1

76 (

16-1

91),

176

(192

-367

), 12

0 (1

389-

1508

), 11

9 (7

17-8

35)

679

(634

-131

2),

367

(74-

440)

, 19

6 (1

313-

1508

), 19

3 (4

41-6

33),

61

(13-

73),

12 (1

-12)

, 119

(717

-835

) 65

8 (5

6-71

3), 3

19 (1

076-

1394

), 31

8 (7

58-1

075)

, 114

(139

5-15

08),

55

(1-5

5), 4

4 (7

14-7

57)

M. e

quig

enita

lium

29

1 (1

039-

1329

), 26

5 (3

68-6

32),

203

(836

-103

8), 1

76 (

16-1

91),

176

(192

-367

), 12

0 (1

389-

1508

), 11

9 (7

17-8

35)

679

(634

-131

2),

367

(74-

440)

, 19

6 (1

313-

1508

), 19

3 (4

41-6

33),

61

(13-

73),

12 (1

-12)

65

8 (5

6-71

3), 3

19 (1

076-

1394

), 31

8 (7

58-1

075)

, 114

(139

5-15

08),

55

(1-5

5), 4

4 (7

14-7

57)

M. e

quir

hini

s 35

4 (1

6-36

9), 3

49 (

370-

718)

, 293

(10

40-1

332)

, 203

(83

7-10

39),

120

(139

2-15

11),

118

(719

-836

), 59

(133

3-13

91)

486

(636

-112

1),

366

(76-

441)

, 19

6 (1

316-

1511

), 19

4 (1

122-

1315

), 17

6 (4

60-6

35),

52 (1

3-64

), 18

(442

-459

), 12

(1-1

2), 1

1 (6

5-75

) 55

8 (1

58-7

15),

435

(107

7-15

11),

237

(840

-107

6),

93 (

65-1

57),

80

(760

-839

), 55

(1-5

5), 4

4 (7

16-7

59),

9 (5

6-64

)

M. f

alco

nis

293

(104

5-13

37),

263

(204

-466

), 25

5 (4

67-7

21),

188

(16-

203)

, 14

7 (8

42-9

88),

120

(722

-841

), 12

0 (1

397-

1516

), 59

(133

8-13

96),

56 (9

89-

1044

), 15

(1-1

5)

561

(78-

638)

, 488

(639

-112

6), 1

96 (1

321-

1516

), 19

4 (1

127-

1320

), 65

(1

3-77

), 12

(1-1

2)

518

(201

-718

), 43

5 (1

082-

1516

), 23

7 (8

45-1

081)

, 82

(76

3-84

4),

55

(1-5

5), 5

5 (5

6-11

0), 4

9 (1

11-1

59),

44 (7

19-7

62),

41 (1

60-2

00)

M. f

astid

iosu

m

329

(274

-602

), 24

0 (6

03-8

42),

184

(132

0-15

03),

147

(843

-989

), 13

0 (1

6-14

5), 1

28 (1

46-2

73),

104

(113

1-12

34),

85 (1

046-

1130

), 85

(123

5-13

19),

56 (9

90-1

045)

, 15

(1-1

5)

493

(143

-635

), 47

9 (8

24-1

302)

, 146

(63

6-78

1), 1

26 (

1303

-142

8), 7

5 (1

429-

1503

), 63

(13

-75)

, 55

(76

-130

), 42

(78

2-82

3),

12 (

1-12

), 12

(131

-142

)

845

(1-8

45),

332

(117

2-15

03),

237

(846

-108

2), 8

9 (1

083-

1171

)

M. f

auci

um

293

(103

7-13

29),

255

(462

-716

), 21

5 (1

6-23

0),

147

(834

-980

), 13

7 (2

31-3

67),

120

(138

9-15

08),

117

(717

-833

), 94

(36

8-46

1), 5

9 (1

330-

1388

), 56

(981

-103

6), 1

5 (1

-15)

485

(634

-111

8), 4

02 (

232-

633)

, 219

(13

-231

), 19

6 (1

313-

1508

), 19

4 (1

119-

1312

), 12

(1-1

2)

556

(158

-713

), 43

5 (1

074-

1508

), 23

7 (8

37-1

073)

, 93

(65

-157

), 70

(7

58-8

27),

55 (1

-55)

, 44

(714

-757

), 9

(56-

64),

9 (8

28-8

36)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. f

elifa

uciu

m

352

(370

-721

), 29

2 (1

044-

1335

), 21

7 (1

6-23

2),

147

(841

-987

), 13

7 (2

33-3

69),

112

(139

5-15

06),

95 (7

22-8

16),

59 (

1336

-139

4), 5

6 (9

88-

1043

), 24

(817

-840

), 15

(1-1

5)

877

(234

-111

0), 2

08 (1

111-

1318

), 18

8 (1

319-

1506

), 16

8 (6

6-23

3), 5

3 (1

3-65

), 12

(1-1

2)

489

(230

-718

), 23

7 (8

44-1

080)

, 187

(12

14-1

400)

, 164

(66

-229

), 13

3 (1

081-

1213

), 10

6 (1

401-

1506

), 81

(763

-843

), 56

(1-5

6), 4

4 (7

19-7

62),

9 (5

7-65

)

M. f

elim

inut

um

966

(294

-125

9), 2

93 (1

-293

), 27

1 (1

260-

1530

) 65

3 (1

-653

), 48

9 (8

45-1

333)

, 197

(133

4-15

30),

191

(654

-844

) 49

1 (2

43-7

33),

318

(778

-109

5),

242

(1-2

42),

234

(109

6-13

29),

143

(138

8-15

30),

58 (1

330-

1387

), 44

(734

-777

)

M. f

elis

28

5 (4

39-7

23),

279

(104

6-13

24),

220

(16-

235)

, 20

3 (2

36-4

38),

147

(843

-989

), 12

2 (1

384-

1505

), 95

(724

-818

), 59

(13

25-1

383)

, 56

(990

-10

45),

24 (8

19-8

42),

15 (1

-15)

446

(668

-111

3), 4

02 (

237-

638)

, 224

(13

-236

), 19

8 (1

308-

1505

), 19

4 (1

114-

1307

), 29

(639

-667

), 12

(1-1

2)

664

(57-

720)

, 318

(765

-108

2), 3

07 (1

083-

1389

), 11

6 (1

390-

1505

), 56

(1

-56)

, 44

(721

-764

)

M. f

erm

enta

ns

382

(339

-720

), 35

0 (1

043-

1392

), 19

4 (1

-194

), 14

7 (8

40-9

86),

112

(139

3-15

04),

107

(232

-338

), 95

(72

1-81

5), 5

6 (9

87-1

042)

, 37

(195

-23

1), 2

4 (8

16-8

39)

681

(636

-131

6),

232

(1-2

32),

212

(319

-530

), 18

8 (1

317-

1504

), 10

5 (5

31-6

35),

86 (2

33-3

18)

507

(211

-717

), 18

7 (1

212-

1398

), 15

0 (8

43-9

92),

145

(66-

210)

, 13

2 (1

080-

1211

), 10

6 (1

399-

1504

), 87

(99

3-10

79),

81 (

762-

842)

, 56

(1-

56),

44 (7

18-7

61),

9 (5

7-65

)

M. f

locc

ular

e 23

4 (3

91-6

24),

206

(106

4-12

69),

202

(862

-106

3), 1

89 (

33-2

21),

179

(135

5-15

33),

169

(222

-390

), 12

0 (7

42-8

61),

117

(625

-741

), 85

(127

0-13

54),

17 (1

6-32

), 15

(1-1

5)

471

(30-

500)

, 29

0 (8

54-1

143)

, 19

6 (1

338-

1533

), 19

4 (1

144-

1337

), 18

6 (5

01-6

86),

167

(687

-853

), 17

(13-

29),

12 (1

-12)

73

8 (1

-738

), 30

1 (1

233-

1533

), 23

6 (8

65-1

100)

, 132

(11

01-1

232)

, 82

(783

-864

), 44

(739

-782

)

M. g

allin

aceu

m

487

(236

-722

), 33

6 (1

045-

1380

), 20

3 (1

-203

), 17

1 (8

18-9

88),

121

(138

1-15

01),

95 (7

23-8

17),

56 (9

89-1

044)

, 32

(204

-235

),

473

(638

-111

0),

401

(237

-637

), 23

6 (1

-236

), 19

7 (1

305-

1501

), 19

4 (1

111-

1304

) 51

9 (2

01-7

19),

305

(108

2-13

86),

237

(845

-108

1), 1

44 (

57-2

00),

115

(138

7-15

01),

81 (7

64-8

44),

56 (1

-56)

, 44

(720

-763

)

M. g

allin

arum

81

6 (1

-816

), 29

1 (1

044-

1334

), 17

1 (8

17-9

87),

112

(139

4-15

05),

59

(133

5-13

93),

56 (9

88-1

043)

68

1 (6

37-1

317)

, 636

(1-6

36),

188

(131

8-15

05)

706

(57-

762)

, 319

(108

1-13

99),

237

(844

-108

0), 1

06 (1

400-

1505

), 81

(7

63-8

43),

56 (1

-56)

M. g

allis

eptic

um

535

(462

-996

), 22

7 (2

35-4

61),

192

(105

3-12

44),

146

(1-1

46),

143

(124

5-13

87),

122

(138

8-15

09),

88 (1

47-2

34),

56 (9

97-1

052)

40

1 (2

36-6

36),

212

(974

-118

5),

186

(788

-973

), 14

6 (6

42-7

87),

131

(1-1

31),

126

(118

6-13

11),

123

(131

2-14

34),

104

(132

-235

), 75

(143

5-15

09),

5 (6

37-6

41)

937

(153

-108

9), 3

02 (1

208-

1509

), 15

2 (1

-152

), 11

8 (1

090-

1207

)

M. g

allo

pavo

nis

489

(232

-720

), 27

7 (1

041-

1317

), 23

1 (1

-231

), 14

7 (8

38-9

84),

121

(137

7-14

97),

95 (

721-

815)

, 59

(131

8-13

76),

56 (

985-

1040

), 22

(816

-83

7)

636

(665

-130

0),

403

(233

-635

), 19

7 (1

301-

1497

), 11

6 (1

-116

), 89

(1

17-2

05),

29 (6

36-6

64),

27 (2

06-2

32)

662

(56-

717)

, 305

(107

8-13

82),

237

(841

-107

7), 1

15 (1

383-

1497

), 79

(7

62-8

40),

55 (1

-55)

, 35

(727

-761

), 9

(718

-726

)

M. g

atea

e 29

3 (1

042-

1334

), 25

5 (4

65-7

19),

203

(839

-104

1),

201

(1-2

01),

169

(202

-370

), 11

9 (7

20-8

38),

96 (

1418

-151

3), 9

4 (3

71-4

64),

59 (

1335

-13

93),

24 (1

394-

1417

)

487

(637

-112

3), 4

29 (2

08-6

36),

196

(131

8-15

13),

194

(112

4-13

17),

131

(77-

207)

, 76

(1-7

6)

558

(159

-716

), 43

5 (1

079-

1513

), 23

7 (8

42-1

078)

, 10

2 (5

7-15

8),

81

(761

-841

), 56

(1-5

6), 4

4 (7

17-7

60)

M. g

enita

lium

24

9 (1

052-

1300

), 23

3 (8

19-1

051)

, 232

(373

-604

), 21

4 (6

05-8

18),

146

(1-1

46),

124

(138

7-15

10),

95 (

278-

372)

, 89

(14

7-23

5),

59 (

1328

-13

86),

42 (2

36-2

77),

27 (1

301-

1327

)

212

(973

-118

4), 2

11 (2

37-4

47),

200

(131

1-15

10),

193

(448

-640

), 15

7 (8

16-9

72),

146

(641

-786

), 13

1 (1

-131

), 12

6 (1

185-

1310

), 93

(14

4-23

6), 2

9 (7

87-8

15),

12 (1

32-1

43)

592

(233

-824

), 30

4 (1

207-

1510

), 26

4 (8

25-1

088)

, 15

2 (1

-152

), 11

8 (1

089-

1206

), 80

(153

-232

)

M. g

lyco

philu

m

488

(235

-722

), 27

7 (1

043-

1319

), 19

4 (1

-194

), 14

7 (8

40-9

86),

121

(137

9-14

99),

95 (

723-

817)

, 59

(132

0-13

78),

56 (

987-

1042

), 32

(203

-23

4), 2

2 (8

18-8

39),

8 (1

95-2

02)

636

(667

-130

2),

402

(236

-637

), 19

7 (1

303-

1499

), 11

7 (1

-117

), 91

(1

18-2

08),

29 (6

38-6

66),

27 (2

09-2

35)

663

(57-

719)

, 305

(108

0-13

84),

237

(843

-107

9), 1

15 (1

385-

1499

), 79

(7

64-8

42),

56 (1

-56)

, 35

(729

-763

), 9

(720

-728

)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. g

ypis

35

2 (1

040-

1391

), 30

1 (6

8-36

8),

255

(463

-717

), 14

7 (8

37-9

83),

120

(139

2-15

11),

119

(718

-836

), 94

(369

-462

), 67

(1-6

7), 5

6 (9

84-1

039)

681

(635

-131

5),

429

(206

-634

), 19

6 (1

316-

1511

), 11

7 (1

-117

), 88

(118

-205

)

714

(1-7

14),

321

(107

7-13

97),

237

(840

-107

6), 1

14 (

1398

-151

1), 8

1

(759

-839

), 44

(715

-758

)

M. h

aem

ocan

is

229

(577

-805

), 21

1 (2

76-4

86),

190

(102

3-12

12),

170

(1-1

70),

167

(129

7-14

63),

147

(820

-966

), 90

(487

-576

), 84

(12

13-1

296)

, 56

(967

-10

22),

46 (1

88-2

33),

42 (2

34-2

75),

17 (1

71-1

87),

14 (8

06-8

19)

667

(613

-127

9),

378

(235

-612

), 23

4 (1

-234

), 10

9 (1

280-

1388

), 75

(1

389-

1463

) 82

9 (2

31-1

059)

, 28

8 (1

176-

1463

), 19

3 (1

-193

), 89

(10

60-1

148)

, 37

(1

94-2

30),

27 (1

149-

1175

)

M. h

aem

ofel

is

229

(577

-805

), 21

1 (2

76-4

86),

190

(102

3-12

12),

170

(1-1

70),

167

(129

7-14

63),

147

(820

-966

), 90

(487

-576

), 84

(12

13-1

296)

, 56

(967

-10

22),

46 (1

88-2

33),

42 (2

34-2

75),

17 (1

71-1

87),

14

(806

-819

)

667

(613

-127

9),

378

(235

-612

), 23

4 (1

-234

), 10

9 (1

280-

1388

), 75

(1

389-

1463

) 82

9 (2

31-1

059)

, 28

8 (1

176-

1463

), 19

3 (1

-193

), 89

(10

60-1

148)

, 37

(1

94-2

30),

27 (1

149-

1175

)

M. h

aem

omur

is

442

(102

2-14

63),

333

(486

-818

), 21

1 (2

75-4

85),

147

(819

-965

), 13

4 (1

-134

), 98

(135

-232

), 56

(966

-102

1), 4

2 (2

33-2

74)

668

(612

-127

9),

378

(234

-611

), 16

6 (6

8-23

3),

109

(128

0-13

88),

75

(138

9-14

63),

67 (1

-67)

12

70 (1

94-1

463)

, 137

(57-

193)

, 56

(1-5

6)

M. h

omin

is

370

(1-3

70),

349

(371

-719

), 29

1 (1

041-

1331

), 14

7 (8

38-9

84),

120

(139

1-15

10),

118

(720

-837

), 59

(133

2-13

90),

56 (9

85-1

040)

55

2 (7

7-62

8), 4

84 (6

37-1

120)

, 196

(131

5-15

10),

194

(112

1-13

14),

76

(1-7

6), 8

(629

-636

) 55

8 (1

59-7

16),

433

(107

8-15

10),

237

(841

-107

7),

93 (

66-1

58),

80

(761

-840

), 56

(1-5

6), 4

4 (7

17-7

60),

9 (5

7-65

)

M. h

yoph

aryn

gis

489

(234

-722

), 29

0 (1

045-

1334

), 19

3 (1

-193

), 11

8 (8

29-9

46),

110

(139

4-15

03),

95 (

723-

817)

, 59

(133

5-13

93),

56 (

989-

1044

), 42

(947

-98

8), 4

0 (1

94-2

33),

11 (8

18-8

28)

680

(638

-131

7), 4

03 (2

35-6

37),

234

(1-2

34),

186

(131

8-15

03)

489

(231

-719

), 31

8 (1

082-

1399

), 23

7 (8

45-1

081)

, 165

(66

-230

), 10

4 (1

400-

1503

), 81

(764

-844

), 56

(1-5

6), 4

4 (7

20-7

63),

9 (5

7-65

)

M. h

yopn

eum

onia

e 23

3 (3

82-6

14),

206

(105

4-12

59),

202

(852

-105

3), 1

80 (

33-2

12),

179

(134

5-15

23),

169

(213

-381

), 12

0 (7

32-8

51),

117

(615

-731

), 85

(126

0-13

44),

32 (1

-32)

,

651

(677

-132

7),

425

(30-

454)

, 20

6 (4

71-6

76),

196

(132

8-15

23),

29

(1-2

9), 1

6 (4

55-4

70)

728

(1-7

28),

318

(773

-109

0), 3

01 (

1223

-152

3), 1

32 (

1091

-122

2), 4

4 (7

29-7

72)

M. h

yorh

inis

29

1 (1

048-

1338

), 23

3 (3

74-6

06),

179

(133

9-15

17),

169

(205

-373

), 14

7 (8

45-9

91),

133

(16-

148)

, 121

(724

-844

), 11

7 (6

07-7

23),

56 (9

92-

1047

), 48

(149

-196

), 15

(1-1

5), 8

(197

-204

)

656

(13-

668)

, 459

(669

-112

7), 1

96 (1

322-

1517

), 19

4 (1

128-

1321

), 12

(1

-12)

72

0 (1

-720

), 43

3 (1

085-

1517

), 23

7 (8

48-1

084)

, 83

(76

5-84

7),

44

(721

-764

)

M. h

yosy

novi

ae

349

(369

-717

), 29

3 (1

039-

1331

), 23

1 (1

-231

), 14

7 (8

36-9

82),

137

(232

-368

), 12

0 (1

391-

1510

), 11

8 (7

18-8

35),

59 (1

332-

1390

), 56

(983

-10

38)

486

(635

-112

0), 4

02 (

233-

634)

, 196

(13

15-1

510)

, 194

(11

21-1

314)

, 15

6 (7

7-23

2), 7

6 (1

-76)

64

9 (6

6-71

4), 4

35 (1

076-

1510

), 23

7 (8

39-1

075)

, 80

(759

-838

), 56

(1-

56),

44 (7

15-7

58),

9 (5

7-65

)

M. i

mita

ns

535

(462

-996

), 22

7 (2

35-4

61),

192

(105

3-12

44),

146

(1-1

46),

143

(124

5-13

87),

122

(138

8-15

09),

88 (1

47-2

34),

56 (9

97-1

052)

40

1 (2

36-6

36),

212

(974

-118

5),

186

(788

-973

), 14

6 (6

42-7

87),

131

(1-1

31),

126

(118

6-13

11),

123

(131

2-14

34),

104

(132

-235

), 75

(143

5-15

09),

5 (6

37-6

41)

937

(153

-108

9), 3

02 (1

208-

1509

), 15

2 (1

-152

), 11

8 (1

090-

1207

)

M. i

ndie

nse

349

(369

-717

), 29

3 (1

039-

1331

), 23

1 (1

-231

), 14

7 (8

36-9

82),

137

(232

-368

), 12

2 (1

391-

1512

), 11

8 (7

18-8

35),

59 (1

332-

1390

), 56

(983

-10

38)

486

(635

-112

0), 4

02 (

233-

634)

, 232

(1-

232)

, 198

(13

15-1

512)

, 19

4 (1

121-

1314

) 55

6 (1

59-7

14),

437

(107

6-15

12),

237

(839

-107

5),

93 (

66-1

58),

80

(759

-838

), 56

(1-5

6), 4

4 (7

15-7

58),

9 (5

7-65

)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. i

ners

49

0 (2

37-7

26),

290

(104

9-13

38),

196

(1-1

96),

147

(846

-992

), 11

2 (1

398-

1509

), 95

(72

7-82

1), 5

9 (1

339-

1397

), 56

(99

3-10

48),

40 (1

97-

236)

, 24

(822

-845

)

680

(642

-132

1), 4

04 (2

38-6

41),

237

(1-2

37),

188

(132

2-15

09)

490

(234

-723

), 31

8 (1

086-

1403

), 23

7 (8

49-1

085)

, 159

(57

-215

), 10

6 (1

404-

1509

), 81

(76

8-84

8), 5

6 (1

-56)

, 44

(724

-767

), 9

(216

-224

), 9

(225

-233

)

M. i

owae

71

9 (2

74-9

92),

249

(113

6-13

84),

144

(1-1

44),

129

(145

-273

), 12

0 (1

385-

1504

), 87

(104

9-11

35),

56 (9

93-1

048)

49

5 (1

42-6

36),

332

(783

-111

4), 1

94 (1

115-

1308

), 14

6 (6

37-7

82),

121

(130

9-14

29),

112

(30-

141)

, 75

(143

0-15

04),

29 (1

-29)

93

5 (1

51-1

085)

, 419

(108

6-15

04),

150

(1-1

50)

M. l

agog

enita

lium

29

1 (1

051-

1341

), 23

3 (3

78-6

10),

203

(848

-105

0), 1

79 (

1342

-152

0),

169

(209

-377

), 15

1 (1

-151

), 12

0 (7

28-8

47),

117

(611

-727

), 57

(15

2-20

8)

672

(1-6

72),

458

(673

-113

0), 1

96 (1

325-

1520

), 19

4 (1

131-

1324

) 51

9 (2

06-7

24),

433

(108

8-15

20),

237

(851

-108

7),

135

(71-

205)

, 82

(7

69-8

50),

70 (1

-70)

, 44

(725

-768

)

M. l

eoni

capt

ivi -

leoc

aptiv

us

706

(16-

721)

, 276

(10

42-1

317)

, 147

(83

9-98

5), 1

21 (

1377

-149

7), 9

5 (7

22-8

16),

59 (1

318-

1376

), 56

(986

-104

1), 2

2 (8

17-8

38),

15(1

-15)

63

5 (6

66-1

300)

, 624

(13-

636)

, 197

(130

1-14

97),

29 (6

37-6

65),

12 (1

-12

) 66

2 (5

7-71

8), 6

20 (

763-

1382

), 11

5 (1

383-

1497

), 56

(1-

56),

35 (

728-

762)

, 9 (7

19-7

27)

M. l

eoph

aryn

gis

447(

373-

819)

, 291

(104

7-13

37),

147(

844-

990)

, 137

(236

-372

), 11

5(16

-13

0),

112(

1397

-150

8),

73(1

31-2

03),

59(1

338-

1396

), 56

(991

-104

6),

32(2

04-2

35),

24(8

20-8

43),

15(1

-15)

681

(640

-132

0),

403

(237

-639

), 22

4 (1

3-23

6),

188

(132

1-15

08),

12

(1-1

2)

498

(233

-730

), 31

9 (1

084-

1402

), 23

7 (8

47-1

083)

, 135

(66

-200

), 10

6 (1

403-

1508

), 81

(76

6-84

6), 5

6 (1

-56)

, 35

(731

-765

), 32

(20

1-23

2), 9

(5

7-65

)

M. l

ipof

acie

ns

291

(104

4-13

34),

266

(370

-635

), 20

0 (3

3-23

2),

147

(841

-987

), 13

7 (2

33-3

69),

112

(139

4-15

05),

95 (

722-

816)

, 86

(636

-721

), 59

(13

35-

1393

), 56

(988

-104

3), 2

4 (8

17-8

40),

17 (1

6-32

), 15

(1-1

5)

681

(637

-131

7),

403

(234

-636

), 20

4 (3

0-23

3),

188

(131

8-15

05),

17

(13-

29),

12 (1

-12)

48

9 (2

30-7

18),

319

(108

1-13

99),

237

(844

-108

0), 1

73 (

57-2

29),

106

(140

0-15

05),

81 (7

63-8

43),

56 (1

-56)

, 44

(719

-762

)

M. l

ipop

hilu

m

290

(104

5-13

34),

230

(235

-464

), 19

4 (1

-194

), 17

2 (4

65-6

36),

147

(842

-988

), 12

8 (1

394-

1521

), 11

9 (7

23-8

41),

86 (

637-

722)

, 59

(133

5-13

93),

56 (9

89-1

044)

, 32

(203

-234

), 8

(195

-202

)

680

(638

-131

7),

235

(1-2

35),

208

(236

-443

), 20

4 (1

318-

1521

), 19

4 (4

44-6

37)

488

(232

-719

), 31

8 (1

082-

1399

), 23

7 (8

45-1

081)

, 166

(66

-231

), 12

2 (1

400-

1521

), 81

(764

-844

), 56

(1-5

6), 4

4 (7

20-7

63),

9 (5

7-65

)

M. m

acul

osum

44

7 (3

73-8

19),

291

(104

7-13

37),

147

(844

-990

), 13

7 (2

36-3

72),

130

(1-1

30),

112

(139

7-15

08),

73 (

131-

203)

, 59

(13

38-1

396)

, 56

(99

1-10

46),

32 (2

04-2

35),

24 (8

20-8

43)

681

(640

-132

0),

403

(237

-639

), 23

6 (1

-236

), 1

88 (1

321-

1508

) 48

9 (2

33-7

21),

319

(108

4-14

02),

237

(847

-108

3), 1

35 (

66-2

00),

106

(140

3-15

08),

81 (

766-

846)

, 56

(1-5

6), 3

5 (7

31-7

65),

32 (

201-

232)

, 9

(57-

65),

9 (7

22-7

30)

M. m

elea

grid

is

489

(235

-723

), 29

1 (1

046-

1336

), 20

2 (1

-202

), 17

1 (8

19-9

89),

112

(139

6-15

07),

95 (

724-

818)

, 59

(133

7-13

95),

56 (

990-

1045

), 32

(203

-23

4)

467

(639

-110

5),

403

(236

-638

), 23

5 (1

-235

), 21

4 (1

106-

1319

), 18

8 (1

320-

1507

) 50

7 (2

14-7

20),

319

(108

3-14

01),

237

(846

-108

2), 1

57 (

57-2

13),

106

(140

2-15

07),

81 (7

65-8

45),

56 (1

-56)

, 35

(730

-764

), 9

(721

-729

)

M. m

icro

ti 32

8 (2

74-6

01),

244

(602

-845

), 14

8 (8

46-9

93),

144

(1-1

44),

144

(124

2-13

85),

129

(145

-273

), 12

1 (1

386-

1506

), 10

5 (1

137-

1241

), 87

(1

050-

1136

), 56

(994

-104

9)

508

(130

-637

), 33

2 (7

84-1

115)

, 194

(111

6-13

09),

146

(638

-783

), 12

2 (1

310-

1431

), 75

(143

2-15

06),

55 (7

5-12

9), 4

5 (3

0-74

), 29

(1-2

9)

698

(151

-848

), 42

0 (1

087-

1506

), 23

8 (8

49-1

086)

, 150

(1-1

50)

M. m

oats

ii 46

8 (3

69-8

36),

291

(104

0-13

30),

180

(133

1-15

10),

147

(837

-983

), 14

5 (1

-145

), 13

7 (2

32-3

68),

86 (1

46-2

31),

56 (9

84-1

039)

30

3 (8

17-1

119)

, 224

(23

3-45

6), 1

97 (

1314

-151

0), 1

94 (

1120

-131

3),

182

(635

-816

), 15

7 (7

6-23

2), 1

27 (4

57-5

83),

75 (1

-75)

, 51

(584

-634

) 75

8 (1

-758

), 43

4 (1

077-

1510

), 23

7 (8

40-1

076)

, 81

(759

-839

)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. m

obile

34

9 (3

68-7

16),

291

(103

9-13

29),

177

(145

-321

), 14

4 (1

-144

), 12

0 (1

389-

1508

), 11

9 (7

17-8

35),

105

(836

-940

), 98

(941

-103

8), 5

9 (1

330-

1388

), 46

(322

-367

)

485

(634

-111

8),

318

(1-3

18),

194

(111

9-13

12),

177

(457

-633

), 12

2 (3

19-4

40),

119

(139

0-15

08),

77 (1

313-

1389

), 16

(441

-456

) 71

3 (1

-713

), 31

8 (7

58-1

075)

, 160

(120

8-13

67),

132

(107

6-12

07),

114

(139

5-15

08),

44 (7

14-7

57),

27 (1

368-

1394

)

M. m

olar

e 47

0 (1

049-

1518

), 23

3 (3

77-6

09),

203

(846

-104

8),

151

(1-1

51),

137

(240

-376

), 11

9 (7

27-8

45),

117

(610

-726

), 56

(152

-207

), 32

(208

-239

) 43

7 (6

72-1

108)

, 43

1 (2

41-6

71),

240

(1-2

40),

196

(132

3-15

18),

194

(112

9-13

22),

20 (1

109-

1128

) 65

3 (7

1-72

3), 4

33 (1

086-

1518

), 23

7 (8

49-1

085)

, 81

(768

-848

), 70

(1-

70),

44 (7

24-7

67)

M. m

uris

77

6 (2

74-1

049)

, 249

(11

37-1

385)

, 144

(1-

144)

, 121

(13

86-1

506)

, 87

(105

0-11

36),

67 (1

45-2

11),

62 (2

12-2

73)

445

(142

-586

), 18

7 (7

84-9

70),

146

(638

-783

), 14

5 (9

71-1

115)

, 12

7 (1

183-

1309

), 12

2 (1

310-

1431

), 75

(14

32-1

506)

, 67

(111

6-11

82),

55

(75-

129)

, 51

(587

-637

), 45

(30-

74),

29 (1

-29)

, 12

(130

-141

)

869

(218

-108

6), 4

20 (1

087-

1506

), 15

0 (1

-150

), 67

(151

-217

)

M. m

uste

lae

488

(234

-721

), 27

6 (1

042-

1317

), 23

3 (1

-233

), 14

7 (8

39-9

85),

121

(137

7-14

97),

95 (

722-

816)

, 59

(131

8-13

76),

56 (

986-

1041

), 22

(817

-83

8)

441

(666

-110

6),

402

(235

-636

), 23

4 (1

-234

), 19

7 (1

301-

1497

), 19

4 (1

107-

1300

), 29

(637

-665

) 66

2 (5

7-71

8), 3

16 (7

63-1

078)

, 304

(107

9-13

82),

115

(138

3-14

97),

56

(1-5

6), 4

4 (7

19-7

62)

M. m

ycoi

des s

sp.

capr

i

236

(605

-840

), 23

4 (1

-234

), 18

6 (2

35-4

20),

184

(421

-604

), 15

7 (9

88-

1144

), 14

7 (8

41-9

87),

105

(114

5-12

49),

99 (

1417

-151

5),

85 (

1250

-13

34),

82 (1

335-

1416

)

378

(260

-637

), 35

2 (7

84-1

135)

, 23

5 (1

-235

), 17

2 (1

146-

1317

), 14

6 (6

38-7

83),

134

(131

8-14

51),

64 (1

452-

1515

), 24

(236

-259

), 10

(113

6-11

45)

717

(1-7

17),

303

(121

3-15

15),

237

(844

-108

0), 1

32 (

1081

-121

2), 8

2 (7

62-8

43),

44 (7

18-7

61)

M. m

ycoi

des s

sp.

myc

oide

s LC

236

(605

-840

), 23

4 (1

-234

), 18

6 (2

35-4

20),

184

(421

-604

), 15

7 (9

88-

1144

), 14

7 (8

41-9

87),

105

(114

5-12

49),

99 (

1417

-151

5),

85 (

1250

-13

34),

82 (1

335-

1416

)

378

(260

-637

), 35

2 (7

84-1

135)

, 23

5 (1

-235

), 17

2 (1

146-

1317

), 14

6 (6

38-7

83),

134

(131

8-14

51),

64 (1

452-

1515

), 24

(236

-259

), 10

(113

6-11

45)

717

(1-7

17),

303

(121

3-15

15),

237

(844

-108

0), 1

32 (

1081

-121

2), 8

2 (7

62-8

43),

44 (7

18-7

61)

M. m

ycoi

des s

sp.

myc

oide

s SC

370

(235

-604

)a , 23

6 (6

05-8

40),

234

(1-2

34),

186

(235

-420

)b , 18

4 (4

21-6

04)b ,

157

(988

-114

4),

147

(841

-987

), 10

5 (1

145-

1249

), 99

(1

417-

1515

), 85

(125

0-13

34),

82 (1

335-

1416

)

378

(260

-637

), 35

2 (7

84-1

135)

, 23

5 (1

-235

), 17

2 (1

146-

1317

), 14

6 (6

38-7

83),

134

(131

8-14

51),

64 (1

452-

1515

), 24

(236

-259

), 10

(113

6-11

45)

717

(1-7

17),

302

(121

3-15

14),

237

(844

-108

0), 1

32 (

1081

-121

2), 8

2 (7

62-8

43),

44 (7

18-7

61)

M. n

euro

lytic

um

291

(105

8-13

48),

247

(1-2

47),

233

(385

-617

), 20

3 (8

55-1

057)

, 17

9 (1

349-

1527

), 13

7 (2

48-3

84),

120

(735

-854

), 11

7 (6

18-7

34)

458

(680

-113

7),

394

(249

-642

), 24

8 (1

-248

), 19

6 (1

332-

1527

), 19

4 (1

138-

1331

), 37

(643

-679

) 73

1 (1

-731

), 43

3 (1

095-

1527

), 23

7 (8

58-1

094)

, 82

(77

6-85

7),

44

(732

-775

)

M. o

pale

scen

s 29

1 (1

044-

1334

), 23

2 (1

-232

), 16

1 (4

75-6

35),

147

(841

-987

), 13

7 (2

33-3

69),

112

(139

4-15

05),

105

(370

-474

), 95

(72

2-81

6), 8

6 (6

36-

721)

, 59

(133

5-13

93),

56 (9

88-1

043)

, 24

(817

-840

)

681

(637

-131

7), 4

03 (2

34-6

36),

233

(1-2

33),

188

(131

8-15

05)

489

(230

-718

), 23

7 (8

44-1

080)

, 187

(12

13-1

399)

, 164

(66

-229

), 13

2 (1

081-

1212

), 10

6 (1

400-

1505

), 81

(763

-843

), 56

(1-5

6), 3

5 (7

28-7

62),

9 (5

7-65

), 9

(719

-727

)

M. o

rale

34

9 (3

69-7

17),

293

(103

9-13

31),

231

(1-2

31),

147

(836

-982

), 13

7 (2

32-3

68),

122

(139

1-15

12),

118

(718

-835

), 59

(133

2-13

90),

56 (9

83-

1038

)

486

(635

-112

0), 4

02 (

233-

634)

, 232

(1-

232)

, 198

(13

15-1

512)

, 19

4 (1

121-

1314

) 55

6 (1

59-7

14),

435

(107

6-15

10),

237

(839

-107

5),

93 (

66-1

58),

80

(759

-838

), 56

(1-5

6), 4

4 (7

15-7

58),

9 (5

7-65

)

M. o

vipn

eum

onia

e 23

3 (3

83-6

15),

206

(105

5-12

60),

202

(853

-105

4), 1

79 (

1346

-152

4),

169

(214

-382

), 14

1 (7

3-21

3),

120

(733

-852

), 11

7 (6

16-7

32),

85

(126

1-13

45),

72 (1

-72)

455

(1-4

55),

290

(845

-113

4),

222

(456

-677

), 19

6 (1

329-

1524

), 19

4 (1

135-

1328

), 16

7 (6

78-8

44)

519

(211

-729

), 31

8 (7

74-1

091)

, 30

1 (1

224-

1524

), 21

0 (1

-210

), 13

2 (1

092-

1223

), 44

(730

-773

)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. o

vis

332

(510

-841

), 27

5 (1

-275

), 21

6 (2

76-4

91),

203

(842

-104

4),

192

(104

5-12

36),

167

(132

2-14

88),

85 (1

237-

1321

), 18

(492

-509

) 46

2 (6

36-1

097)

, 368

(79

-446

), 18

9 (4

47-6

35),

112

(111

2-12

23),

109

(130

5-14

13),

81 (

1224

-130

4),

78 (

1-78

), 75

(14

14-1

488)

, 14

(109

8-11

11)

501

(259

-759

), 31

9 (8

54-1

172)

, 31

6 (1

173-

1488

), 16

0 (9

9-25

8),

98

(1-9

8), 9

4 (7

60-8

53)

M. o

xoni

ensi

s 46

8 (3

71-8

38),

277

(104

2-13

18),

233

(1-2

33),

147

(839

-985

), 13

7 (2

34-3

70),

121

(137

8-14

98),

59 (1

319-

1377

), 56

(986

-104

1)

484

(637

-112

0), 4

02 (

235-

636)

, 197

(13

02-1

498)

, 181

(11

21-1

301)

, 11

8 (1

-118

), 89

(119

-207

), 27

(208

-234

) 53

2 (2

31-7

62),

305

(107

9-13

83),

237

(842

-107

8), 1

74 (

57-2

30),

115

(138

4-14

98),

79 (7

63-8

41),

56 (1

-56)

M. p

enet

rans

392

(599

-990

), 32

5 (2

74-5

98),

167

(123

9-14

05),

144

(1-1

44),

129

(145

-273

), 98

(140

6-15

03),

87 (1

047-

1133

), 84

(115

5-12

38),

56 (9

91-

1046

), 21

(113

4-11

54)

493

(142

-634

), 33

2 (7

81-1

112)

, 194

(111

3-13

06),

146

(635

-780

), 12

1 (1

307-

1427

), 11

2 (3

0-14

1), 7

6 (1

428-

1503

), 29

(1-2

9)

933

(151

-108

3), 4

20 (1

084-

1503

), 15

0 (1

-150

)

M. p

hoci

cere

bral

e 29

3 (1

044-

1336

), 25

5 (4

67-7

21),

195

(1-1

95),

169

(204

-372

), 14

7 (8

41-9

87),

120

(139

6-15

15),

119

(722

-840

), 94

(37

3-46

6), 5

9 (1

337-

1395

), 56

(988

-104

3), 8

(196

-203

)

560

(79-

638)

, 487

(639

-112

5), 1

96 (1

320-

1515

), 19

4 (1

126-

1319

), 78

(1

-78)

55

8 (1

61-7

18),

435

(108

1-15

15),

237

(844

-108

0),

104

(57-

160)

, 81

(7

63-8

43),

56 (1

-56)

, 44

(719

-762

)

M. p

hoci

dae/

phoc

ae

293

(104

4-13

36),

255

(467

-721

), 19

5 (1

-195

), 16

9 (2

04-3

72),

147

(841

-987

), 12

0 (1

396-

1515

), 11

9 (7

22-8

40),

94 (

373-

466)

, 59

(133

7-13

95),

56 (9

88-1

043)

, 8(1

96-2

03)

509

(79-

587)

, 487

(639

-112

5), 1

96 (1

320-

1515

), 19

4 (1

126-

1319

), 78

(1

-78)

, 51

(588

-638

) 55

8 (1

61-7

18),

435

(108

1-15

15),

237

(844

-108

0),

104

(57-

160)

, 81

(7

63-8

43),

56 (1

-56)

, 44

(719

-762

)

M. p

hoci

rhin

is

489

(233

-721

), 29

1 (1

044-

1334

), 14

7 (8

41-9

87),

141

(1-1

41),

112

(139

4-15

05),

95 (

722-

816)

, 91

(142

-232

), 59

(13

35-1

393)

, 56

(988

-10

43),

24 (8

17-8

40)

681

(637

-131

7), 4

03 (2

34-6

36),

233

(1-2

33),

188

(131

8-15

05)

489

(230

-718

), 31

8 (7

63-1

080)

, 187

(12

13-1

399)

, 155

(57

-211

), 13

2 (1

081-

1212

), 10

6 (1

400-

1505

), 56

(1-5

6), 4

4 (7

19-7

62),

9 (2

12-2

20),

9 (2

21-2

29)

M. p

irum

61

3 (2

35-8

47),

192

(105

2-12

43),

148

(848

-995

), 14

6 (1

-146

), 12

3 (1

387-

1509

), 88

(147

-234

), 84

(124

4-13

27),

59 (1

328-

1386

), 56

(996

-10

51)

211

(236

-446

), 18

9 (4

47-6

35),

186

(787

-972

), 14

6 (6

41-7

86),

145

(973

-111

7),

131

(1-1

31),

126

(118

5-13

10),

123

(131

1-14

33),

104

(132

-235

), 76

(143

4-15

09),

67 (1

118-

1184

), 5(

636-

640)

936

(153

-108

8), 3

03 (1

207-

1509

), 15

2 (1

-152

), 11

8 (1

089-

1206

)

M. p

neum

onia

e

233

(819

-105

1), 2

32 (3

73-6

04),

214

(605

-818

), 17

8 (1

052-

1229

), 14

6 (1

-146

), 12

2 (1

387-

1508

), 98

(12

30-1

327)

, 95

(27

8-37

2),

89 (

147-

235)

, 59

(132

8-13

86),

42 (2

36-2

77)

225

(237

-461

), 19

8 (1

311-

1508

), 17

9 (4

62-6

40),

157

(816

-972

), 14

6 (6

41-7

86),

145

(973

-111

7), 1

31 (

1-13

1), 1

26 (

1185

-131

0), 9

3 (1

44-

236)

, 67

(111

8-11

84),

29 (7

87-8

15),

12 (1

32-1

43)

592

(233

-824

), 30

2 (1

207-

1508

), 26

4 (8

25-1

088)

, 15

2 (1

-152

), 11

8 (1

089-

1206

), 80

(153

-232

)

M. p

rim

atum

48

9 (2

34-7

22),

291

(104

5-13

35),

233

(1-2

33),

147

(842

-988

), 11

9 (7

23-8

41),

112

(139

5-15

06),

59 (1

336-

1394

), 56

(989

-104

4)

1318

(1-1

318)

, 188

(131

9-15

06)

489

(231

-719

), 31

9 (1

082-

1400

), 23

7 (8

45-1

081)

, 165

(66

-230

), 10

6 (1

401-

1506

), 81

(76

4-84

4),

56 (

1-56

), 35

(72

9-76

3),

9 (5

7-65

), 9

(720

-728

)

M. p

ullo

rum

33

4 (9

82-1

315)

, 27

4 (2

33-5

06),

232

(1-2

32),

210

(507

-716

), 17

0 (8

12-9

81),

120

(137

5-14

94),

95 (7

17-8

11),

59 (1

316-

1374

) 66

7 (6

32-1

298)

, 42

5 (2

07-6

31),

180

(131

5-14

94),

134

(1-1

34),

72

(135

-206

), 15

(129

9-13

13),

1(13

14-1

314)

65

7 (5

7-71

3), 3

06 (1

075-

1380

), 23

8 (8

37-1

074)

, 114

(138

1-14

94),

79

(758

-836

), 56

(1-5

6), 4

4 (7

14-7

57)

M. p

ulm

onis

29

0 (1

048-

1337

), 27

8 (4

47-7

24),

238

(1-2

38),

166

(281

-446

), 14

7 (8

45-9

91),

96 (

1422

-151

7), 9

5 (7

25-8

19),

59 (

1338

-139

6), 5

6 (9

92-

1047

), 42

(239

-280

), 25

(820

-844

), 25

(139

7-14

21)

484

(837

-132

0),

394

(240

-633

), 23

9 (1

-239

), 19

7 (1

321-

1517

), 16

6 (6

71-8

36),

29 (6

42-6

70),

8 (6

34-6

41)

665

(57-

721)

, 23

7 (8

48-1

084)

, 18

7 (1

216-

1402

), 13

1 (1

085-

1215

), 11

5 (1

403-

1517

), 82

(766

-847

), 56

(1-5

6), 4

4 (7

22-7

65)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. p

utre

faci

ens

236

(605

-840

), 23

4 (1

-234

), 18

6 (2

35-4

20),

184

(421

-604

), 15

7 (9

88-

1144

), 14

7 (8

41-9

87),

105

(114

5-12

49),

99 (

1417

-151

5),

85 (

1250

-13

34),

82 (1

335-

1416

)

378

(260

-637

), 32

3 (8

13-1

135)

, 23

5 (1

-235

), 17

2 (1

146-

1317

), 14

6 (6

38-7

83),

134

(131

8-14

51),

64 (1

452-

1515

), 29

(78

4-81

2), 2

4 (2

36-

259)

, 10(

1136

-114

5)

717

(1-7

17),

237

(844

-108

0), 2

18 (

1213

-143

0), 1

32 (

1081

-121

2), 8

5 (1

431-

1515

), 82

(762

-843

), 44

(718

-761

)

M. s

aliv

ariu

m

350

(372

-721

), 29

3 (1

043-

1335

), 20

2 (1

-202

), 14

7 (8

40-9

86),

137

(235

-371

), 12

1 (1

395-

1515

), 11

8 (7

22-8

39),

59 (1

336-

1394

), 56

(987

-10

42),

32 (2

03-2

34)

486

(639

-112

4),

403

(236

-638

), 23

5 (1

-235

), 19

7 (1

319-

1515

), 19

4 (1

125-

1318

) 51

9 (2

00-7

18),

436

(108

0-15

15),

237

(843

-107

9),

93 (

66-1

58),

80

(763

-842

), 56

(1-5

6), 4

4 (7

19-7

62),

41 (1

59-1

99),

9 (5

7-65

)

M. s

imba

e

489

(233

-721

), 30

7 (9

88-1

294)

, 23

2 (1

-232

), 14

7 (8

41-9

87),

112

(139

6-15

07),

95 (7

22-8

16),

59 (1

337-

1395

), 42

(129

5-13

36),

24 (8

17-

840)

683

(637

-131

9), 4

03 (2

34-6

36),

233

(1-2

33),

188

(132

0-15

07),

48

9 (2

30-7

18),

370

(844

-121

3), 1

55 (

57-2

11),

110

(129

2-14

01),

106

(140

2-15

07),

81 (

763-

843)

, 78

(121

4-12

91),

56 (1

-56)

, 44

(719

-762

), 9

(212

-220

), 9

(221

-229

)

M. s

p. b

ovin

e

grou

p 7

236

(605

-840

), 23

4 (1

-234

), 18

6 (2

35-4

20),

184

(421

-604

), 18

1 (1

335-

1515

)a , 15

7 (9

88-1

144)

, 14

7 (8

41-9

87),

105

(114

5-12

49),

99

(141

7-15

15)b , 8

5 (1

250-

1334

), 82

(133

5-14

16)b

378

(260

-637

), 35

2 (7

84-1

135)

, 23

5 (1

-235

), 17

2 (1

146-

1317

), 14

6 (6

38-7

83),

134

(131

8-14

51),

64 (1

452-

1515

), 24

(236

-259

), 10

(113

6-11

45)

717

(1-7

17),

303

(121

3-15

15),

237

(844

-108

0), 1

32 (

1081

-121

2), 8

2 (7

62-8

43),

44 (7

18-7

61)

M. s

perm

atop

hilu

m

291

(104

2-13

32),

276

(444

-719

), 23

0 (1

-230

), 21

3 (2

31-4

43),

203

(839

-104

1), 1

12 (1

392-

1503

), 95

(720

-814

), 59

(133

3-13

91),

24 (8

15-

838)

487

(635

-112

1),

403

(232

-634

), 23

1 (1

-231

), 19

4 (1

122-

1315

), 18

8 (1

316-

1503

) 60

9 (1

08-7

16),

319

(107

9-13

97),

237

(842

-107

8), 1

06 (

1398

-150

3),

81 (7

61-8

41),

56 (1

-56)

, 51

(57-

107)

, 44

(717

-760

)

M. s

pum

ans

375

(1-3

75),

293

(104

7-13

39),

255

(470

-724

), 14

7 (8

44-9

90),

120

(139

9-15

18),

119

(725

-843

), 94

(376

-469

), 59

(13

40-1

398)

, 56

(991

-10

46)

561

(81-

641)

, 487

(642

-112

8), 1

96 (1

323-

1518

), 19

4 (1

129-

1322

), 80

(1

-80)

51

8 (2

04-7

21),

270

(108

4-13

53),

237

(847

-108

3), 1

65 (

1354

-151

8),

106

(57-

162)

, 81

(766

-846

), 56

(1-5

6), 4

4 (7

22-7

65),

41 (1

63-2

03)

M. s

turn

idae

33

3 (1

043-

1375

), 26

5 (3

70-6

34),

232

(1-2

32),

205

(635

-839

), 14

7 (8

40-9

86),

137

(233

-369

), 12

1 (1

376-

1496

), 56

(987

-104

2)

473

(636

-110

8),

402

(234

-635

), 23

3 (1

-233

), 19

5 (1

302-

1496

), 19

3 (1

109-

1301

) 41

7 (1

080-

1496

), 36

3 (4

00-7

62),

343

(57-

399)

, 31

7 (7

63-1

079)

, 56

(1

-56)

M. s

ualv

i 46

8 (3

69-8

36),

291

(104

0-13

30),

179

(133

1-15

09),

147

(837

-983

), 14

5 (1

-145

), 13

7 (2

32-3

68),

86 (1

46-2

31),

56 (9

84-1

039)

35

1 (2

33-5

83),

303

(817

-111

9), 1

94 (1

120-

1313

), 18

2 (6

35-8

16),

157

(76-

232)

, 99

(141

1-15

09),

97 (1

314-

1410

), 75

(1-7

5), 5

1 (5

84-6

34)

702

(57-

758)

, 433

(107

7-15

09),

237

(840

-107

6), 8

1 (7

59-8

39),

56 (1

-56

)

M. s

ubdo

lum

29

3 (1

040-

1332

), 25

5 (4

63-7

17),

231

(1-2

31),

147

(837

-983

), 13

7 (2

32-3

68),

120

(139

2-15

11),

119

(718

-836

), 94

(36

9-46

2), 5

9 (1

333-

1391

), 56

(984

-103

9)

487

(635

-112

1),

402

(233

-634

), 23

2 (1

-232

), 19

6 (1

316-

1511

), 19

4(11

22-1

315)

55

6 (1

59-7

14),

435

(107

7-15

11),

237

(840

-107

6),

93 (

66-1

58),

81

(759

-839

), 56

(1-5

6), 4

4 (7

15-7

58),

9 (5

7-65

)

M. s

uis

333

(524

-856

), 28

9 (1

-289

), 27

7 (1

060-

1336

), 21

6 (2

90-5

05),

203

(857

-105

9), 1

67 (1

337-

1503

), 18

(506

-523

) 64

9 (1

-649

), 50

0 (6

50-1

149)

, 10

9 (1

320-

1428

), 89

(11

50-1

238)

, 81

(1

239-

1319

), 75

(142

9-15

03)

501

(273

-773

), 41

4 (7

74-1

187)

, 316

(118

8-15

03),

272

(1-2

72)

M. s

ynov

iae

371

(1-3

71),

277

(104

4-13

20),

265

(723

-987

), 14

0 (4

65-6

04),

120

(138

0-14

99),

118

(605

-722

), 93

(372

-464

), 59

(13

21-1

379)

, 56

(988

-10

43)

489

(815

-130

3),

309

(1-3

09),

196

(130

4-14

99),

194

(444

-637

), 17

7 (6

38-8

14),

134

(310

-443

) 66

3 (5

7-71

9), 3

05 (1

081-

1385

), 23

8 (8

43-1

080)

, 114

(138

6-14

99),

79

(764

-842

), 56

(1-5

6), 4

4 (7

20-7

63)

M. t

estu

dine

um -

chel

onia

e

371

(1-3

71),

277

(104

4-13

20),

265

(723

-987

), 14

0 (4

65-6

04),

120

(138

0-14

99),

118

(605

-722

), 93

(372

-464

), 59

(13

21-1

379)

, 56

(988

-10

43)

489

(815

-130

3),

309

(1-3

09),

196

(130

4-14

99),

194

(444

-637

), 17

7 (6

38-8

14),

134(

310-

443)

66

0 (5

7-71

6), 3

00 (1

226-

1525

), 23

7 (8

57-1

093)

, 132

(109

4-12

25),

82

(775

-856

), 58

(717

-774

), 56

(1-5

6)


R

estri

ctio

n en

donu

clea

ses

Myc

opla

sma

spp.

AluI

Bf

aI

Hpy

F10V

I (M

woI

)

M. t

estu

dini

s 76

0 (2

35-9

94),

193

(105

3-12

45),

146

(1-1

46),

122

(138

9-15

10),

88

(147

-234

), 84

(124

6-13

29),

59 (1

330-

1388

), 58

(995

-105

2)

228

(236

-463

), 18

6 (7

86-9

71),

171

(464

-634

), 14

6 (6

40-7

85),

135

(972

-110

6),

131

(1-1

31),

126

(118

7-13

12),

123

(131

3-14

35),

104

(132

-235

), 75

(14

36-1

510)

, 67

(112

0-11

86),

13 (

1107

-111

9), 5

(63

5-63

9)

937

(153

-108

9), 3

02 (1

209-

1510

), 15

2 (1

-152

), 11

9 (1

090-

1208

)

M. v

erec

undu

m

583

(237

-819

), 27

9 (1

045-

1323

), 20

4 (1

-204

), 14

7 (8

42-9

88),

121

(138

3-15

03),

59 (

1324

-138

2), 5

6 (9

89-1

044)

, 32

(205

-236

), 22

(820

-84

1)

667

(640

-130

6), 4

02 (2

38-6

39),

237

(1-2

37),

197

(130

7-15

03)

709

(57-

765)

, 307

(108

2-13

88),

237

(845

-108

1), 1

15 (1

389-

1503

), 79

(7

66-8

44),

56 (1

-56)

M. w

enyo

nii

332

(506

-837

), 27

1 (1

-271

), 21

6 (2

72-4

87),

192

(104

1-12

32),

187

(131

8-15

04),

147

(838

-984

), 85

(123

3-13

17),

56 (9

85-1

040)

, 18

(488

-50

5)

364

(79-

442)

, 272

(82

2-10

93),

189

(443

-631

), 11

2 (1

108-

1219

), 10

2 (7

20-8

21),

95 (

1410

-150

4), 8

8 (6

32-7

19),

83 (

1301

-138

3), 8

1 (1

220-

1300

), 78

(1-7

8), 2

6 (1

384-

1409

), 8

(109

4-11

01),

6 (1

102-

1107

)

501

(255

-755

), 33

6 (1

169-

1504

), 31

9 (8

50-1

168)

, 25

4 (1

-254

), 94

(7

56-8

49)

M. y

eats

ii 23

7 (6

05-8

41),

234

(1-2

34),

186

(235

-420

), 18

4 (4

21-6

04),

157

(989

-11

45),

147

(842

-988

), 10

5 (1

146-

1250

), 99

(14

18-1

516)

, 85

(12

51-

1335

), 82

(133

6-14

17)

378

(260

-637

), 32

4 (8

13-1

136)

, 23

5 (1

-235

), 19

8 (1

319-

1516

), 17

2 (1

147-

1318

), 14

6 (6

38-7

83),

29 (

784-

812)

, 24

(236

-259

), 10

(11

37-

1146

)

717

(1-7

17),

237

(845

-108

1), 2

18 (

1214

-143

1), 1

32 (

1082

-121

3), 8

5 (1

432-

1516

), 83

(762

-844

), 44

(718

-761

)

1 Myc

opla

sma

spec

ies

with

a re

vise

d ta

xano

my

(i.e.

M. l

actu

cae,

M. s

omni

lux,

M. m

elal

euca

e, M

. lum

inos

um, M

. luc

ivor

ax, a

nd M

. elly

chni

ae) a

re n

ot in

clud

ed, w

hile

the

6 m

embe

rs o

f

the

M. m

ycoi

des c

lust

er (i

.e. M

. cap

rico

lum

sspp

., M

. myc

oide

s ssp

p., a

nd M

. sp.

bov

ine

grou

p 7)

are

. 2 If

no

rest

rictio

n pa

ttern

is m

arke

d bo

ld, o

ther

rest

rictio

n en

zym

es (a

s sug

gest

ed in

the

man

uscr

ipt)

are

need

ed to

diff

eren

tiate

this

spec

ies.

a, b in

dica

te p

ossi

ble

diff

eren

ces b

etw

een

rrnA

(a) a

nd rr

nB (b

)



Discussion Identification of mycoplasmas still largely relies on serological tests, but owing to the limited

availability of quality-controlled sera, the high number of species, the serological cross-

reaction between related species and the great variability in the surface antigens of different

strains (36), newer techniques are needed. Sequence analysis of the 16S rRNA genes proved a

useful tool to identify species, but the need for expensive equipment makes the technique less

favorable for routine diagnosis. In this study, we showed that theoretically all Mycoplasma

spp. are distinguishable using ARDRA. The in silico determined discriminative power was

confirmed in the laboratory and even closely related Mycoplasma spp. could be identified

correctly, as exemplified by the restriction with AluI and BfaI of M. agalactiae and M. bovis.

We used universal primers to amplify the entire 16S rDNA to obtain a maximum

discriminatory power. Working with universal primers implies that interference from other

bacteria is to be expected when starting from clinical samples (9), especially when

mycoplasmas are not abundantly present. The use of mycoplasma-specific primers binding to

internal regions of the 16S rRNA genes may be helpful and result in a higher specificity as

was already proposed by others (4, 12). However, care must be taken since the discriminatory

power will decrease if primers are chosen in such a way that less restriction sites are present

in the amplification products. Alternatively, McAuliffe et al. (28) proposed a selective

enrichment step for 24 hours in Eaton's-medium before amplification of 16S sequences to

identify Mycoplasma spp. Also Kiss et al. (22) used ARDRA to identify three avian

Mycoplasma species after 48 hours of incubation in Frey media. These suggested approaches

may solve most problems, but may still be insufficient for mixed Mycoplasma cultures. The

presence of more than one Mycoplasma species in clinical samples will lead to complex

patterns, which are not easily resolved.

Differences between rrn operons have been reported in several bacterial classes, but the level

of sequence heterogeneity was recently shown to be lower than expected (1). It is therefore

reasonable to assume that rrn operons tend to evolve in concert (25). For some bacterial

species a high level of 16S rDNA sequence heterogeneity has been described (24, 29), while

for Mycoplasma species, which possess no more than 2 rrn operons, only some micro-

heterogeneity (i.e. scattered sequence variation between highly related rRNA genes) has been

reported (2, 21, 32). Besides, most differences between the two operons will not lead to

altered restriction sites and will not influence the ARDRA patterns. In case a mutation is


located within one of both restriction recognition sites, as was shown in particular for

M. columbinum, restriction will most likely yield an unknown ARDRA profile, rather than

lead to a false identification. Moreover, this aberrant pattern can be included in the

identification scheme. The significance of the C1007T transition (E. coli numbering) present

in two of the four M. columbinum strains is still unknown, but was shown in some strains of

E. coli as well (25). Also, in agreement with an earlier report (32), many differences between

the rrnA and rrnB sequences were observed for members of the M. mycoides cluster.

Nevertheless, the combined restriction profiles of both rrn sequences resulted in expected

patterns with exception of a faint band seen for M. capricolum subsp. capripneumoniae after

restriction with HpyF10VI. The reason for this partial restriction is unknown since purifying

the PCR product, increasing the enzyme concentration, or lengthening the incubation period

made no difference (data not shown). In any case, identification based on ARDRA was shown

complex for these very related species and other techniques – like serological tests

independent of the 16S rDNA sequences (7) - may be more suitable. However, the extra band

visible for M. mycoides subsp. mycoides SC after restriction with AluI was shown sufficiently

stable to be used for identification (31) and the value of ARDRA using PstI was also reported

for M. capricolum subsp. capripneumoniae (2). Although the 16S rDNA sequences of these

species may be almost identical, ARDRA is able to emphasise the few differences present

without the need of extensive 16S rDNA sequence analysis or other tests (6, 31, 35, 39). Also

for other species with nearly identical 16S rDNA sequences (99.5% identity for M.

haemocanis and M. haemofelis; 99.7% for M. gallisepticum and M. imitans; 98.9% for M.

orale and M. indiense, and 99.8% for M. criteculi and M. collis), it was calculated that

restriction analysis with a single additional enzyme would result in different restriction

patterns and therefore to a correct identification.

In conclusion, restriction digestion with AluI of the amplified 16S rDNA can be used to

differentiate between 73 of the 116 described Mycoplasma species and subspecies. An

additional restriction with BfaI or HpyF10VI enables the identification of another 31 species

and subspecies. Also the remaining 12 species can be differentiated, with the use of additonal

enzymes, although other techniques may be preferred for some members of the M. mycoides-

cluster.

The simplicity and the general applicability of ARDRA make it possible to implement this

technique in most laboratories with basic molecular biology equipment.


Acknowledgements This work was supported by a grant of the Federal Service of Public Health, Food Chain

Safety and Environment (Grant number S-6136).

The authors thank Sara Tistaert for skilful technical assistance.

References

1. Acinas, S. G., L. A. Marcelino, V. Klepac-Ceraj, and M. F. Polz. 2004. Divergence and redundancy

of 16S rRNA sequences in genomes with multiple rrn operons. J Bacteriol. 186:2629-35.

2. Bascunana, C. R., J. G. Mattsson, G. Bolske, and K. E. Johansson. 1994. Characterization of the

16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based

on PCR. J. Bacteriol. 176:2577-2586.

3. Bencina, D., and J. M. Bradbury. 1992. Combination of immunofluorescence and immunoperoxidase

techniques for serotyping mixtures of Mycoplasma species. J. Clin. Microbiol. 30:407-410.

4. Blanchard, A., M. Gautier, and V. Mayau. 1991. Detection and identification of mycoplasmas by

amplification of rDNA. FEMS Microbiol. Lett. 65:37-42.

5. Bolske, G. 1988. Survey of Mycoplasma infections in cell cultures and a comparison of detection

methods. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A]. 269:331-340.

6. Bolske, G., J. G. Mattsson, C. R. Bascunana, K. Bergstrom, H. Wesonga, and K. E. Johansson.

1996. Diagnosis of contagious caprine pleuropneumonia by detection and identification of Mycoplasma

capricolum subsp. capripneumoniae by PCR and restriction enzyme analysis. J Clin Microbiol. 34:785-

791.



8. Chalker, V. J., W. M. Owen, C. J. Paterson, and J. Brownlie. 2004. Development of a polymerase

chain reaction for the detection of Mycoplasma felis in domestic cats. Vet. Microbiol. 100:77-82.

9. Conrads, G., T. F. Flemmig, I. Seyfarth, F. Lampert, and R. Lutticken. 1999. Simultaneous

detection of Bacteroides forsythus and Prevotella intermedia by 16S rRNA gene-directed multiplex

PCR. J. Clin. Microbiol. 37:1621-1624.

10. Criado-Fornelio, A., A. Martinez-Marcos, A. Buling-Sarana, and J. C. Barba-Carretero. 2003.

Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from

southern Europe: a molecular study. Vet. Microbiol. 93:307-317.

11. Daffonchio, D., A. Cherif, L. Brusetti, A. Rizzi, D. Mora, A. Boudabous, and S. Borin. 2003.

Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of

Bacillus and related genera. Appl. Environ. Microbiol. 69:5128-5137.





13. Djordjevic, S. P., G. J. Eamens, L. F. Romalis, and M. M. Saunders. 1994. An improved enzyme

linked immunosorbent assay (ELISA) for the detection of porcine serum antibodies against

Mycoplasma hyopneumoniae. Vet. Microbiol. 39:261-273.

14. Edwards, U., T. Rogall, H. Blocker, M. Emde, and E. C. Bottger. 1989. Isolation and direct

complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal

RNA. Nucleic Acids Res. 17:7843-7853.

15. Erno, H., and K. Peterslund. 1983. Growth precipitation test, p. 489-492. In S. Razin, and J. G. Tully

(ed.), Methods in Mycoplasmology: Mycoplasma Characterization, vol. I. Academic Press.

16. Fan, H. H., S. H. Kleven, M. W. Jackwood, K. E. Johansson, B. Pettersson, and S. Levisohn. 1995.

Species identification of avian mycoplasmas by polymerase chain reaction and restriction fragment

length polymorphism analysis. Avian Dis. 39:398-407.

17. Friis, N. F., P. Ahrens, and H. Larsen. 1991. Mycoplasma hyosynoviae isolation from the upper

respiratory tract and tonsils of pigs. Acta Vet. Scand. 32:425-429.

18. Garcia, M., M. W. Jackwood, S. Levisohn, and S. H. Kleven. 1995. Detection of Mycoplasma

gallisepticum, M. synoviae, and M. iowae by multi-species polymerase chain reaction and restriction

fragment length polymorphism. Avian Dis. 39:606-616.

19. Harasawa, R., and Y. Kanamoto. 1999. Differentiation of two biovars of Ureaplasma urealyticum

based on the 16S-23S rRNA intergenic spacer region. J. Clin. Microbiol. 37:4135-4138.

20. Harasawa, R., H. Mizusawa, K. Nozawa, T. Nakagawa, K. Asada, and I. Kato. 1993. Detection and

tentative identification of dominant Mycoplasma species in cell cultures by restriction analysis of the

16S-23S rRNA intergenic spacer regions. Res. Microbiol. 144:489-493.

21. Heldtander, M., B. Pettersson, J. G. Tully, and K. E. Johansson. 1998. Sequences of the 16S rRNA

genes and phylogeny of the goat mycoplasmas Mycoplasma adleri, Mycoplasma auris, Mycoplasma

cottewii and Mycoplasma yeatsii. Int. J. Syst. Bacteriol. 48 Pt 1:263-268.

22. Kiss, I., K. Matiz, E. Kaszanyitzky, Y. Chavez, and K. E. Johansson. 1997. Detection and

identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length

polymorphism assay. Vet. Microbiol. 58:23-30.

23. Kobisch, M., and N. F. Friis. 1996. Swine mycoplasmoses. Rev. Sci. Tech. 15:1569-1605.

24. Marchandin, H., C. Teyssier, M. Simeon De Buochberg, H. Jean-Pierre, C. Carriere, and E.

Jumas-Bilak. 2003. Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the

genus Veillonella: implications for phylogeny and taxonomy. Microbiology. 149:1493-1501.


25. Martinez-Murcia, A. J., A. I. Anton, and F. Rodriguez-Valera. 1999. Patterns of sequence variation

in two regions of the 16S rRNA multigene family of Escherichia coli. Int. J. Syst. Bacteriol. 49 Pt

2:601-610.

26. May, J. D., and S. L. Branton. 1997. Identification of Mycoplasma isolates by ELISA. Avian Dis.

41:93-96.



Microbiol. 41:4844-4847.

28. McAuliffe, L., F. M. Hatchell, R. D. Ayling, A. I. King, and R. A. Nicholas. 2003. Detection of

Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in

England. Vet. Rec. 153:687-688.

29. Mevarech, M., S. Hirsch-Twizer, S. Goldman, E. Yakobson, H. Eisenberg, and P. P. Dennis. 1989.

Isolation and characterization of the rRNA gene clusters of Halobacterium marismortui. J. Bacteriol.

171:3479-3485.

30. Miles, R., and R. Nicholas. 1998. Mycoplasma protocols, vol. 104. Coordinating ed., J. M. Walker.

Humana Press, Totowa, New Jersy.




3821.



genes from the two rRNA operons. J. Bacteriol. 178:4131-4142.

33. Rawadi, G., and O. Dussurget. 1998. Genotypic methods for diagnosis of mycoplasmal infections in

humans, animals, plants and cell cultures. Biotechnol. Genet. Eng. Rev. 15:51-78.

34. Razin, S., and J. G. Tully. 1983. Methods in Mycoplasmology, vol. I. Academic Press.

35. Rodriguez, J. L., R. W. Ermel, T. P. Kenny, D. L. Brooks, and A. J. DaMassa. 1997. Polymerase

chain reaction and restriction endonuclease digestion for selected members of the "Mycoplasma

mycoides cluster" and Mycoplasma putrefaciens. J Vet Diagn Invest. 9:186-190.

36. Rosengarten, R., and D. Yogev. 1996. Variant colony surface antigenic phenotypes within

mycoplasma strain populations: implications for species identification and strain standardization. J.


37. Saglio, P. H. M., and R. F. Whitcomb. 1979. Diversity of wall-less prokaryotes in plant vascular

tissue, fungi, and invertebrate animals, p. 1-31. In M. F. Barile, S. Razin, R. F. Whitcomb, and J. G.

Tully (ed.), The mycoplasmas: plant and insects mycoplasmas, vol. III. Academic Press, Inc., London.


38. Thomas, C. B., and P. Sharp. 1988. Detection of antigenic variation among strains of Mycoplasma

gallisepticum by enzyme-linked immunosorbent inhibition assay (ELISIA) and Western blot analysis.

Avian Dis. 32:748-756.

39. Vilei, E. M., and J. Frey. 2004. Differential clustering of Mycoplasma mycoides subsp. mycoides SC

strains by PCR-REA of the bgl locus. Vet. Microbiol. 100:283-288.

40. Woese, C. R., J. Maniloff, and L. B. Zablen. 1980. Phylogenetic analysis of the mycoplasmas. Proc.

Natl. Acad. Sci. USA. 77:494-498.

Evaluation of tDNA-PCR for the identification of Mollicutes 101

III.2 EVALUATION OF TDNA-PCR FOR THE IDENTIFICATION

OF MOLLICUTES

Tim Stakenborg1, Jo Vicca2, Rita Verhelst3, Patrick Butaye1, Dominiek Maes2, Anne

Naessens4, Geert Claeys3, Catharine De Ganck3, Freddy Haesebrouck2, and Mario

Vaneechoutte3


Belgium 3 Department of Clinical Chemistry, Microbiology & Immunology, Ghent University

Hospital, De Pintelaan 185, 9000 Ghent, Belgium 4 Department of Microbiology, University of Brussels (VUB) Hospital, Laarbeeklaan 101,

1090 Brussels, Belgium

Published in: Journal of Clinical Microbiology (2005) 43(9):4558-4566.

102 Evaluation of tDNA-PCR for the identification of Mollicutes

Abstract We evaluated the applicability of tDNA-PCR in combination with fluorescent capillary

electrophoresis on an ABI310 genetic analyzer (Applied Biosystems, Ca.) for the

identification of different mollicute species. A total of 103 strains and DNA extracts of 30

different species belonging to the genera Acholeplasma, Mycoplasma and Ureaplasma were

studied. Reproducible peak profiles were generated for all samples, except for one M.

genitalium, the three M. gallisepticum isolates and eight of the 24 Ureaplasma cultures, where

no amplification could be obtained. Clustering revealed numerous discrepancies compared to

the identifications that had been previously obtained by means of biochemical and serological

tests. Final identification was obtained by 16S rRNA gene amplification followed by

sequence analysis and/or restriction digestion (ARDRA). This confirmed in all cases the

identification obtained by tDNA-PCR. Seven samples yielded an unexpected tDNA-PCR

profile. Sequence analysis of the 16S rDNA showed that six of these samples were mixed and

one had a unique sequence that did not match with any of the published sequences, pointing

to the existence of a not yet described species. In conclusion, we found tDNA-PCR to be a

rapid and discriminatory method to correctly identify a large collection of different species of

the class of Mollicutes and to recognise not yet described groups.

Introduction Having no cell wall, Mollicutes form a special class of bacteria. Their small, compact

genomes evolved from AT-rich, gram-positive bacteria by means of genome reduction. At the

same time, they developed innovative mechanisms to survive as parasitic organisms in a wide

variety of host environments. To date, eight genera belonging to the class of Mollicutes have

been described and within these genera up to 200 species, mostly of the genus Mycoplasma,

are acknowledged. This variety of species is associated with several taxonomic unclarities

(15, 17, 18) and a correct identification may be found very difficult for numerous reasons.

Firstly, a number of Mollicutes, especially the plant-pathogenic spiroplasmas, have not been

cultivated, while others require very complex media. As a result, only a limited number of

isolates exist for some species and these are often not easily accessible. Secondly, as more

sequences and better isolation media become available, more species are continuously being

discovered. Finally, for some species limited data and very few reports are published,

especially for low- and non-virulent species.


To correctly differentiate all these species, a universal and fast identification technique would

be extremely useful. Some promising methods have already been described (e.g. 25), but they

do not yield digitised data, making exchange between laboratories difficult. An optimised

tDNA-PCR technique, originally described by Welsh and McClelland (27, 48), has been

shown useful to correctly and reproducibly identify very diverse bacterial species when

combined with high resolution electrophoresis (3-5, 10, 11, 23, 45). The technique is based on

the amplification of spacer regions in between tRNA genes using consensus tDNA primers.

The amplified products are separated by electrophoresis, for exact sizing, and the resulting

species-specific peak profiles are subsequently archived in a database. Profiles obtained from

an unknown sample can be compared with this data set while not yet included and/or newly

described species can be added to expand the database further. We investigated the potential

of this technique to correctly identify a large number of Acholeplasma, Mycoplasma, and

Ureaplasma species.


Strains

A total of 103 strains and DNA extracts were used during this study and are listed in Table 1.

Purified genomic DNA of isolates belonging to the M. mycoides cluster was kindly supplied

by Dr. L. Manso-Silivan (CIRAD, France) and of M. hyosynoviae isolates by Dr. B.

Kokotovic (DFVF, Denmark). The DNA extracts from clinical samples positive for

M. genitalium were received from the Institute of Tropical Diseases (Antwerp, Belgium) and

had been extracted directly from vaginal swabs of five Asian women. Apart from the

reference strains included, all isolates were obtained over the years during routine diagnostics.

The origin of these strains was not always clear since some strains were retrieved from old

collections.

Culture media and DNA extraction

F-broth (7), A7 differential agar (37), modified Hayflick broth (MHB) (44), SP-4-broth (44),

SP-4-broth supplemented with L-arginine (SP4A), HS-broth (16), or Friis’-broth with

ampicillin instead of methicillin (NHS20) (21) were used to cultivate the different species, as

listed in Table 1. Genomic DNA was prepared from the cultivated strains and from the

vaginal swabs by means of phenol/chloroform extraction as described before (28), except for


the U. urealyticum, U. parvum, M. hominis and M. salivarium strains for which DNA was

extracted by alkaline lysis (46).

Identification of strains

Most strains (i.e. MYP10-58 and MYP65-74) had been identified previously by means of

phenotypical characteristics and the growth precipitation test using absorbed rabbit antisera

(13, 36). After identification, the strains had been lyophilised in the presence of 20% sterile

milk and stored at 4°C. U. urealyticum, U. parvum and some of the M. hominis isolates were

previously identified by their characteristic growth on A7 differential agar medium and by

their ability to hydrolyze urea and arginine, respectively. Due to the numerous discrepancies

with the results obtained in this study by means of tDNA-PCR, most of the strains were re-

identified. The identity of M. hyopneumoniae, M. hyorhinis and M. flocculare was confirmed

by a specific PCR (40). Also the M. genitalium samples were identified by two specific PCR

tests as described by Jensen et al. (19, 20). Most other isolates were re-identified using

amplified rDNA restriction analysis (ARDRA) (III.1) and/or sequence analysis of the 16S

rDNA (12, 46) (see Table 1).

tDNA-PCR and cluster analysis

tDNA-PCR was performed as described previously using primers T5A

(5’AGTCCGGTGCTCTAACCAACTGAG) and primer T3B (5’

AGGTCGCGGGTTCGAATCC) (4, 27). Cluster analysis of the obtained tDNA-PCR

fingerprints was carried out by calculating a distance matrix using the differential base pair

(dbp) algorithm (3) with a tolerance of 1.2 bp and including all peaks (i.e. no noise

subtraction) from 50 to 500 base pairs in length. The dbp clustering algorithm was used to

calculate the similarity by taking the average of the two results that are obtained by dividing

the number of tDNA-intergenic spacers in common between two strains by the total number

of spacers of one strain, respectively of the other strain. A similarity tree was constructed

using the UPGMA method (PHYLIP, V3.6, Felsenstein, J., Department of Genome Sciences,

University of Washington, Seattle, Wa.) and visualised using Treeview V1.6.6 (29).


Table 1: Overview of the isolates used in this studya.

Received as Final Identification tDNA-PCR based ID Isolated/ Date of Strain name Culture (Techniqueb) identification Received Isolation medium

fromc (mm/yy) Avian mollicutes

M. columbinasale M. columbinasale (A) M. columbinasale MYP030 CODA 04/89 397 HS

M. columbinum M. columbinum (S) M. columbinum MYP031 CODA 12/87 446 HS

M. columbinum M. columbinum (S) M. columbinum MYP032 CODA 11/85 423VD HS

M. columbinum M. columbinum (S) M. columbinum MYP033 CODA 12/87 447 HS

M. gallinaceum M. gallinarum (S) M. gallinarum MYP038 CODA NAd CODA 18A HS

M. gallisepticum M. gallinarum (A) M. gallinarum MYP041 CODA 01/89 D63P F

M. gallisepticum M. gallinarum (A) M. gallinarum MYP042 CODA 02/89 CODA 19E F

M. gallisepticum M. gallisepticum (A, S) No peaks MYP013 CODA NA ATCC 19610 MHB

M. gallisepticum Mixed profile (S) Mixed profile MYP039 CODA 04/89 X95 HS

M. gallisepticum M. gallisepticum (S) No peaks MYP040 CODA 04/89 A5969 F

M. gallisepticum M. gallisepticum (A) No peaks MYP071 CODA NA 2000Myc58 F

M. glycophilum M. glycophilum (A) M. glycophilum MYP043 CODA 02/89 CODA 20A MHB

M. lipofaciens M. lipofaciens (A) M. lipofaciens MYP049 CODA 01/88 R171 MHB

M. pullorum M. glycophilum (A) M. glycophilum MYP052 CODA 12/84 412VD F

M. pullorum M. columborale (S) M. columborale MYP053 CODA 12/86 Pul46 MHB

M. synoviae M. neurolyticum (A) M. neurolyticum MYP058 CODA 02/69 WVU1853 HS

Bovine, caprine and ovine mollicutes

M. agalactiae M. arginini (A) M. arginini MYP016 CODA 06/84 884/200 HS

M. agalactiae M. agalactiae (A) M. bovis-agalactiae MYP017 CODA NA NCTC 10123 (PG2) HS

M. agalactiae M. bovis (A) M. bovis-agalactiae MYP018 CODA 04/97 83/61 HS

M. agalactiae M. agalactiae (A) M. bovis-agalactiae MYP019 CODA NA 5725 HS

M. bovigenitalium M. bovigenitalium (S) M. bovigenitalium MYP020 CODA 06/89 MN120 MHB

M. bovirhinis M. bovirhinis (A) M. bovirhinis MYP066 CODA NA ATCC 27748 NHS20

M. bovis M. bovis (A) M. bovis-agalactiae MYP022 CODA 06/83 295VD F

M. bovis M. bovis (A) M. bovis-agalactiae MYP023 CODA NA Widanka309 F

M. bovis M. bovis (S) M. bovis-agalactiae MYP067 CODA NA O422 MHB

M. bovis M. bovirhinis (A) M. bovirhinis MYP068 CODA NA O475 MHB

M. dispar M. bovis (A) M. bovis-agalactiae MYP034 CODA 11/83 CODA 17A SP4

M. dispar M. dispar (A) M. dispar MYP035 CODA 12/82 CODA 17B SP4

M. dispar M. dispar (A) M. dispar MYP036 CODA NA ATCC 27140 SP4

M. dispar M. bovis (A, S) M. bovis-agalactiae MYP037 CODA 11/83 CODA 17E SP4

M. capricolum ssp. M. capricolum ssp.. M. capricolum MYP080 CIRAD NA ATCC 27343 NA capricolum capricolum (A) (California Kid)



fromc (mm/yy)

M. capricolum ssp. M. capricolum ssp. M. capricolum MYP076 CIRAD NA NCTC 10192 NA capripneumoniae capripneumoniae (S) (F38)

M. mycoides ssp. M. mycoides bsp. M. mycoides MYP078 CIRAD NA Pg3 NA capri capri (A)

M. mycoides ssp. M. mycoides ssp. M. mycoides MYP079 CIRAD NA YG NA mycoides LC mycoides LC (A)

M. mycoides ssp. M. mycoides ssp. M. mycoides ssp. MYP075 CODA NA Pg1 NA mycoides SC mycoides SC (A) mycoides SC

M. sp. bovine M. sp. bovine M. sp. bovine MYP077 CIRAD NA Pg50 NA group 7 group 7 (A) group 7

M. ovipneumoniae M. bovis (S) M. bovis-agalactiae MYP051 CODA 08/83 CODA 29C F

M. putrefaciens M. putrefaciens (A) M. putrefaciens MYP054 CODA 11/85 Put85 F

M. putrefaciens M. putrefaciens (A) M. putrefaciens MYP055 CODA 03/87 B387 F

M. putrefaciens M. putrefaciens (A) M. putrefaciens MYP056 CODA 03/87 B791 F

M. putrefaciens M. putrefaciens (A) M. putrefaciens MYP057 CODA 02/98 7578.95 F

Human mollicutes

M. genitalium M. genitalium (P) M. genitalium A MYP106 ITG NA MSE 0883 NA

M. genitalium M. genitalium (P) No peaks MYP107 ITG NA MSE 0896 NA

M. genitalium M. genitalium (P) M. genitalium B MYP108 ITG NA MSE 1028 NA

M. genitalium M. genitalium (P) M. genitalium A MYP109 ITG NA MSE 1209 NA

M. genitalium M. genitalium (P) M. genitalium B MYP110 ITG NA MSE 1318 NA

M. hominis Mixed profile (NT) Mixed profile MYP081 VUB 03/04 040319/5 A7

M. hominis M. hominis (S) M. hominis MYP111 GUH 02/02 020211 2245 A7

M. hominis M. hominis (S) M. hominis MYP112 GUH NA BVS058A4 A7

M. orale M. orale (S) M. orale MYP115 NCTC NA ATCC 23714 SP4A

M. pneumoniae M. pneumoniae (A) M. pneumoniae MYP072 CODA 06/96 CODA 38B SP4

M. pneumoniae M. pneumoniae (A) M. pneumoniae MYP073 CODA 12/85 CODA 38C SP4

M. pneumoniae M. pneumoniae (A) M. pneumoniae MYP074 CODA 12/84 CODA 38D SP4

M. salivarium M. salivarium (S) M. salivarium MYP113 GUH NA ED135 SP4A

M. salivarium M. salivarium (S) M. salivarium MYP114 GUH NA TC010 SP4A

U. urealyticum & Mixed profile (NT) Mixed profile MYP082 VUB NA 040324/3 A7 M. hominis

U. urealyticum & Mixed profile (NT) Mixed profile MYP083 VUB 10/03 031002/9 A7 M. hominis U. urealyticum & NA No peaks MYP084 VUB 04/04 040413/1 A7 M. hominis

U. parvum NA U. parvum MYP085 VUB NA Serotype 01 A7

U. urealyticum NA No peaks MYP086 VUB NA Serotype 02 A7

U. parvum NA No peaks MYP087 VUB NA Serotype 03 A7



fromc (mm/yy)

U. urealyticum NA U. urealyticum MYP088 VUB NA Serotype 04 A7











U. urealyticum NA No peaks MYP099 VUB 03/04 040327/1 A7

U. urealyticum NA U. urealyticum MYP100 VUB 03/04 040323/1 A7



U. urealyticum NA U. parvum MYP103 VUB 03/04 040329/7 A7

U. urealyticum & NA Mixed profile MYP104 VUB 10/03 031001/3 A7 M. hominis

U. urealyticum & NA Mixed profile MYP105 VUB 03/04 040324/3 A7 M. hominis

Murine mollicutes

M. neurolyticum M. neurolyticum (A) M. neurolyticum MYP050 CODA 01/75 CODA 28A HS

Porcine mollicutes

A. granularum A. granularum (A) A. granularum MYP015 CODA 02/77 CODA 2D MHB

M. flocculare M. flocculare (P) M. flocculare MYP001 CODA NA MP102 NHS20

M. flocculare M. flocculare (P) M. flocculare MYP002 CODA NA ATCC 27399 NHS20 (Ms42)

M. flocculare M. flocculare (P) M. flocculare MYP003 CODA 07/00 MflocF6A NHS20

M. hyopneumoniae M. hyopneumoniae M. hyopneumoniae MYP007 CODA NA ATCC 25934 (J) NHS20 (A, P)

M. hyopneumoniae M. hyopneumoniae M. hyopneumoniae MYP008 CODA 12/99 MhF5C NHS20

M. hyopneumoniae M. hyopneumoniae M. hyopneumoniae MYP009 CODA 07/00 MhF6A NHS20 (A, P)

M. hyopneumoniae M. hyorhinis (P) M. hyorhinis MYP044 CODA 12/83 Pf NHS20

M. hyopneumoniae A. laidlawii (A) A. laidlawii A MYP045 CODA 03/83 RotaRA NHS20

M. hyorhinis M. hyorhinis (P) M. hyorhinis MYP004 CODA 07/00 MhyorF7A NHS20





fromc (mm/yy)

M. hyosynoviae M. hyosynoviae (A) M. hyosynoviae MYP059 DFVF NA Mp6 NA





M. hyosynoviae M. hyosynoviae (A) M. hyosynoviae MYP064 CODA NA ATCC 25591(S16) NA

Other mollicutes

A. laidlawii A. laidlawii (A) A. laidlawii A MYP010 CODA 03/84 84/DAW MHB

A. laidlawii A. laidlawii (A) A. laidlawii B MYP011 CODA 08/87 87/328VD MHB

A. laidlawii A. laidlawii (A) A. laidlawii B MYP012 CODA 07/82 CODA 1E MHB

A. laidlawii A. sp. nov (S) A. sp. nov MYP014 CODA 08/85 CODA 1G MHB

A. laidlawii A. laidlawii (A) A. laidlawii B MYP065 CODA NA ATCC 23206 NHS20 (PG8) a: The samples are listed according their host and in alphabetical order as received (and discussed in the results section). b: A: ARDRA, P: species specific PCR, S: 16S rRNA gene sequence analysis, NT: not tested. c: CODA = Veterinary and Agrochemical Research Centre (Brussels, Belgium); ITG = Institute of Tropical Diseases

(Antwerp, Belgium); GUH = Ghent University Hospital (Belgium); VUB = Free University of Brussels (Belgium);

DFVF = Danish Institute for Food and Veterinary Research (Copenhagen, Denmark), CIRAD = Agricultural Research

Centre for International Development (Montpellier, France), NCTC = National Collection of Type Cultures (London,

UK). d: NA = not applicable/not available.

Construction of a digital library

A digital library composed of consensus library entries was constructed. Each consensus

library entry contained only peaks (amplified tDNA intergenic spacers) present in all sample

files of a particular species. Identification was carried out by comparing the fingerprint of a

strain with all entries of the constructed library using the dbp algorithm. For identification,

dbp takes into account only peaks that are mutually present in the sample file and in the

library entry, discarding the peaks only present in the sample file. For example, comparison of

an unknown with 15 peaks, of which 10 are identical to all 10 peaks of a library entry, gives a

similarity of 100%, as would have been the case for an unknown with 10 peaks, all identical

to the 10 peaks of the library entry. An unknown with 8 peaks of which all 8 are identical to 8

of the 10 peaks of the library entry gives a value of only 80%. An unknown with 15 peaks of

which 8 are identical to an entry with 10 peaks gives an identity of 80% as well. This method

is (by experience) better suited for identification of unknown patterns, since intraspecific


variability of the peaks with low intensity, is better compensated for. To differentiate between

resembling, but distinct tDNA PCR patterns, the algorithm allows to take absent peaks into

account by adding a minus in front of the absent peak. The software counts the absence of the

peak as a positive match, increasing the similarity score and increasing the reliability of the

identification result.

Results

Cluster analysis of tDNA-PCR fingerprints

tDNA-PCR fingerprints could be obtained from 91 of the 103 strains and DNA-extracts in

total. In general, strains showed fingerprints with more than 10 amplified DNA fragments, i.e.

intergenic tRNA-spacer regions. In most cases the obtained tDNA-PCR-patterns were species

specific and highly identical for all the strains tested of a single species. The consensus library

entries for each species are listed in Table 2. Below we present the clustering results (shown

in Figure 1) obtained for the different species, grouped according to their host.

Avian mollicutes

The one strain (MYP30) received as M. columbinasale, of which the identification was

confirmed by ARDRA, clustered separately, most closely to the M. gallinarum cluster.

The three strains MYP31-MYP33 received as M. columbinum had identical patterns,

clustering separately.

One strain received as M. gallinaceum (MYP38) and two of the six strains received as M.

gallisepticum (MYP41 and MYP42) had an identical, unique tDNA-PCR pattern and were

identified by ARDRA as M. gallinarum.

Six strains (MYP13, MYP39 – MYP42 and MYP71) were received as M. gallisepticum. As

explained, MYP41 and MYP42 were shown to be M. gallinarum. No amplification products

were obtained from strains MYP13, MYP40 and MYP71, identified as M. gallisepticum

according to ARDRA or sequence analysis. The tDNA-PCR pattern of strain MYP39 showed

a high similarity to members of the M. bovis – M. agalactiae cluster, although the tDNA-

pattern profile had clearly many additional peaks. Sequence analysis showed a mixed profile,

confirming the contamination of this sample, which was therefore left out for further analysis.

The M. glycophilum strain (MYP43), of which the identification was confirmed by ARDRA,

was indistinguishable from strain MYP52, received as M. pullorum, which was identified by

ARDRA as M. glycophilum.


The one M. lipofaciens strain (MYP49), confirmed as such by ARDRA, had a very specific

tDNA-PCR pattern.

Two strains (MYP52 and MYP53) were received as M. pullorum. One (MYP52) was

identified as M. glycophilum by ARDRA as discussed above. The other one (MYP53) was

identified by sequence analysis as M. columborale, a species that was not included initially,

which is consistent with the fact that this strain had a unique tDNA-PCR pattern.

The one strain received as M. synoviae (MYP58) had a tDNA-PCR pattern that strongly

resembled that of the sole M. neurolyticum strain (MYP50), an identification that was

confirmed by ARDRA.

Bovine, caprine and ovine mollicutes

Four strains (MYP16-19) were received as M. agalactiae. The DNA-extract obtained from M.

agalactiae strain MYP16 was found to yield a separate tDNA-PCR-fingerprint (Figure 1).

This strain was later identified with ARDRA as M. arginini. The two genuine M. agalactiae

strains (MYP17 & MYP19) had a very similar tDNA profile and clustered together with strain

MYP18, which was later identified as M. bovis by means of ARDRA.

The M. bovigenitalium strain MYP20 had a unique tDNA-PCR pattern and its identification

could be confirmed by 16S rDNA sequence analysis.

Strains MYP22-23 and MYP67-68 had previously been identified as M. bovis. MYP68

clustered together with MYP66, received as M. bovirhinis, and was shown by ARDRA to be

indeed M. bovirhinis. The remaining three M. bovis strains clustered together with the M.

agalactiae strains MYP17 and MYP19.

Four strains were received as M. dispar (MYP34-37). MYP34 and MYP37 were clustered by

tDNA-PCR in the M. bovis - M. agalactiae group, a finding substantiated by ARDRA, which

identified both strains as M. bovis. This identification was confirmed for MYP37 by

sequencing. The remaining M. dispar strains MYP35 and MYP36 clustered separately (Figure

1) and were shown to be genuine M. dispar by ARDRA.

The so-called M. mycoides cluster comprises six species or subspecies of closely related

mycoplasmas. The type strains of these ruminant mycoplasmas were included for analysis

(MYP75 – MYP80). M. capricolum subsp. capricolum (MYP80) and in particular, M.

capricolum subsp. capripneumoniae (MYP76) clustered most closely to the Mycoplasma sp.

bovine group 7 strain (MYP77). These profiles showed up to 30 peaks, with over 20 peaks in

common. Likewise, the tDNA profiles of the three M. mycoides isolates showed several small


peaks and clustered together, with M. mycoides subsp. capri (MYP78) and M. mycoides

subsp. mycoides LC (MYP79) most closely related to each other.

The one strain received as M. ovipneumoniae (MYP51) clustered within the M. bovis-M.

agalactiae group (Figure 1) and was identified as M. bovis by 16S rDNA sequence analysis.

The four M. putrefaciens strains (MYP54 - MYP57), confirmed as such by ARDRA, had a

very identical and characteristic tDNA-PCR pattern.

Human mollicutes

Five samples were received as M. genitalium (MYP106 - MYP110). Strain MYP107 did not

yield a fingerprint. Strikingly, there were two different groups observed within M. genitalium

(see Table 2), which did cluster separately, but close to each other.

Three M. hominis cultures were included in this study. Two pure M. hominis isolates

(MYP111 and MYP112), identified by means of 16S rDNA sequencing, had identical and

specific tDNA-PCR patterns composed of only two peaks (of 151.9 and 226.6 bp). One

culture (MYP081) positive for M. hominis on A7 agar plates, was clearly contaminated, since

additional peaks of 56.0, 144.2 and 280.7 bp - shown in this study to be characteristic for

U. urealyticum - were present. Only the tDNA-PCR fingerprints of the pure isolates MYP111

and MYP112 were included in the cluster analysis.

One M. orale (MYP115) was obtained from a culture collection (National Collection of Type

Cultures, UK) and had a very specific tDNA-PCR profile with over 25 characteristic peaks.

The tDNA-PCR patterns of the three strains received as M. pneumoniae (MYP72 – MYP74),

confirmed as such by ARDRA, were almost identical and were characterised by very short

spacers (usually no longer than 77 bp).

The two M. salivarium strains (MYP113 and MYP114) had been isolated during studies of

the complex microflora of tonsils and teeth and their identity had been established by 16S

rRNA sequencing. Their nearly identical and highly characteristic tDNA-patterns clustered

together.

A total of 24 Ureaplasma strains, received as U. urealyticum or U. parvum (MYP82-

MYP105), were included. For eight strains, no amplification could be obtained. Of the

remaining samples, four (MYP82-MYP83 and MYP104-MYP105) had been received as

contaminated with M. hominis, which was also apparent – as explained above when

presenting the results for M. hominis - from the mixed tDNA-PCR pattern that was obtained

and that contained peaks characteristic to both species. These tDNA-PCR fingerprints were


not included in the cluster analysis. All other strains of both Ureaplasma species gave similar

tDNA-PCR patterns and clustered together.

Murine mollicutes

The one strain (MYP50) that was received as M. neurolyticum and confirmed as such by

ARDRA, had a tDNA-PCR pattern that clustered together with that of strain MYP58,

received as M. synoviae, but shown to be M. neurolyticum, as described above.

Porcine mollicutes

The one strain received as A. granularum (MYP15) was confirmed as such by ARDRA and

could be identified easily by tDNA-PCR, since its pattern was highly characteristic.

The three M. flocculare strains (MYP1 - MYP3) had a very similar pattern that made it

possible to differentiate this species from all other species.

Five strains (MYP7 - MYP9, MYP44 and MYP45) were received as M. hyopneumoniae. A

specific PCR identified MYP44 as M. hyorhinis, while MP45 was identified as A. laidlawii as

described below. MYP7, MYP8, and MYP9 had identical and characteristic tDNA-PCR

fingerprints, and their identity as M. hyopneumoniae was confirmed by ARDRA and by a

specific PCR.

The strains MYP4 - MYP6 received as M. hyorhinis, were very much alike and had a typical

pattern. The genuine M. hyorhinis strains clustered together with MYP44, which had been

shown to be M. hyorhinis as well (see above).

Six strains (MYP59 – MYP64), received as M. hyosynoviae and confirmed as such by

ARDRA, were very much alike with regard to their characteristic tDNA-PCR pattern.

Other mollicutes

Five strains (MYP10 - MYP12, MYP14 and MYP65) were received as A. laidlawii. MYP14

had a unique tDNA-PCR pattern and clustered separately. Sequence analysis showed

significant differences with other known Acholeplasma spp. and the 16S rRNA gene sequence

was submitted to Genbank (Accession Number AY785356). The other four strains clustered

together with strain MYP45, which was received as M. hyopneumoniae, but also identified as

A. laidlawii by means of ARDRA.

tDNA-PCR based identification

A digital library was constructed as described. For A. laidlawii and M. genitalium, different

tDNA-PCR profiles were apparent and for these species, two different consensus patterns


were included in the library. All individual fingerprints (sample files including all peaks) were

compared with this library using the similarity calculation designated dbp. The two

M. agalactiae strains were indistinguishable from the eight M. bovis strains, having tRNA-

spacers with lengths of 57.4, 61.5, 67.3, 70.0, 78.6, 131.4, 144.2, 151.8, 159.5 and 257.4 bp in

common. All other samples were identified correctly. Although the U. urealyticum and U.

parvum strains grouped together during cluster analysis, they could clearly be distinghuished

on the basis of their specific tDNA-PCR pattern. The strains MYP85 (serovar 1), MYP90

(serovar 6) and MYP98 (serovar 14) belong to U. parvum. These three stains, together with

MYP103, for which serovar determination had not been carried out, had a peak of 279.5 bp

(standard deviation 0.1) in common, whereas the U. urealyticum strains MYP88 (serovar 4),

MYP89 (serovar 5), MYP91 (serovar 7), MYP93 (serovar 9), MYP94 (serovar 10), MYP95

(serovar 11), MYP96 (serovar 12) and MYP100 (serovar not determined) had a peak of 280.7

bp (standard deviation 0.03 bp) in common.


Table 2: Consensus tDNA-PCR profiles of tested Mollicutes species.

Species1 Number Consensus tDNA-PCR fragments (in bp)2

Acholeplasma laidlawii A 2 58.6; 62.2; 66.7; 69.1; 70.9; 156.6; 189.3; 281.1; 389.6

Acholeplasma laidlawii B 3 58.6; 62.2; 66.7; 70.9; 72.3; 191.3; 281.1; 391.6

Acholeplasma granularum 1 57.5; 61.4; 70; 178.2; 270; 385.2; 463.5

Acholeplasma sp. nov 1 57.6; 62.4; 66.5; 68.7; 70.9; 283.5; 303.2; 352.8; 433.3

M. bovis – M. agalactiae 7 - 2 57.4; 61.5; 67.3; 70; 78.6; 131.4; 144.2; 151.8; 159.5; 257.4

M. arginini 1 56.9; 64.4; 86.4; 132.3; 144.1; 152.6; 161.1; 197.5; 224.2; 227.7

M. bovigenitalium 1 59.9; 67.6; 76.6; 88.8; 132.6; 146.4; 153.2; 161.1; 358.1

M. bovirhinis 2 55.3; 61.3; 63.6; 67.7; 68.7; 69.7; 76.9; 79.2; 135.6; 140.1; 141.8; 147.2; 150; 157.3

M. capricolum ssp. 2 55.3; 70; 124.5; 132.1; 135.5; 138.3; 140.6; 142.3; 143.9; 145; 147.1; 156.7; 158.1; 212.9; 225.6; 226.3; 228.6; 235.4; 241.3; 245.5 M. columbinasale 1 57.4; 59.9; 65.2; 67.7; 76.6; 85.9; 91.8; 132; 146.1; 152.8; 160.3; 256.6; 349.9

M. columbinum 3 57.3; 60.6; 66.2; 69; 77.7; 130.5; 143.6; 148.1; 150.4; 158.3; 222.5; 257.2; 350.3

M. columborale 1 57.2; 59.4; 67.6; 74.9; 77.1; 133.4; 146.9; 154.2; 247.6

M. dispar 2 69.7; 131; 150.6; 287.6

M. flocculare 3 128.7; 148.3; 158.2; 291.3

M. gallinarum 3 57; 64.5; 66.4; 75.9; 85.8; 113.1; 131; 144.7; 151.5; 160.1; 256.6; 347; 349.9

M. genitalium A 2 56.4; 66.2; 145.6; 181.2

M. genitalium B 2 58.8; 64.4; 66.5; 76.6; 88.3; 125.6; 143.6; 154.5; 177.2; 198.5; 217.6; 228.5

M. glycophilum 2 58.5; 66.7; 74.3; 76.5; 133.2; 147; 154.2; 358.2

M. hominis 2 151.9; 226.7

M. hyopneumoniae 3 69.2; 132.6; 153.8; 311.1

M. hyorhinis 4 59.3; 67.3; 144.6; 150; 230; 245.3

M. hyosynoviae 6 57.1; 66.1; 129.5; 149.2; 153.1; 198.2; 228.7; 244.9; 262.3; 322

M. lipofaciens 1 56.4; 57.7; 62.2; 64.2; 65.8; 74.9; 85.3; 127.1; 139.8; 146.3; 152.8

M. mycoides ssp. mycoides LC & 2 55.3; 72.1; 134.8; 155.4; 211.5; 223.8; 226.8; 233.4; 237.7; 239.2; 243.5 M. mycoides ssp. capri M. mycoides ssp. mycoides SC 1 55.3; 69.4; 72.4; 156.4; 159.9; 212; 216.2; 218.6; 224.5; 232.4; 234.4; 236.3; 238.7; 240.3; 244.6 M. neurolyticum 2 63.5; 73.2; 126.7; 139.9; 145.7; 168.4; 230.5

M. orale 1 56.5; 59.4; 65.6; 66.7; 67.6; 89.8; 118; 131.3; 134.3; 135.6; 137.5; 139.6; 147.7; 150; 151.4; 153; 161.1; 173.1; 199.1; 221.8; 225.8; 229.7; 238.3; 241.6; 256.7; 323.7 M. pneumoniae 3 55.3; 59.1; 61.4; 65.8; 67.2; 70.8; 73.6; 75.1; 77.2

M. putrefaciens 4 60.3; 68.8; 134; 149.5; 154.8; 165; 210.3; 225.2; 241.6; 249.4

M. salivarium 2 64.6; 66.4; 131.9; 152.1; 229.3; 258.2; 320.9

Mycoplasma sp. bovine group 7 1 55.3; 72.2; 73.2; 106.2; 121.6; 124.5; 131.1; 132.1; 135.5; 138.3; 139.4; 140.6; 141.7; 142.6; 144.1; 145; 147.2; 156.7; 158; 212.2; 215.8; 216.5; 224.8; 227.6; 232.7; 234.6; 236.2; 238.8; 240.2; 244.5 Ureaplasma urealyticum 8 56; 144.2; -279.5; 280.7

Ureaplasma parvum 4 55.4; 144.2; 279.5; -280.7 1 Species that were indistinguishable are listed together 2 A minus (-) preceding a number indicates the absence of a peak with that length in bp.


Figure 1: Dendrogram of tDNA-PCR fingerprints obtained after cluster analysis with

UPGMA of dbp based similarity coefficients. Strains are listed with the final identifications

obtained.


Discussion The combination of biochemical and serological results has always been a valuable tool for

the identification of mollicutes. However, biochemical data often lack discriminatory power,

while also the problem of serological cross-reaction has been described (6, 34, 35, 41). The

problems with serological identification are exemplified in this study by the fact that 14 out of

53 serologically characterised strains had been misidentified. In this study, we evaluated

whether a genotypic identification method, like tDNA-PCR, might increase the efficiency of

identification.

In general, over 10 different peaks were visible in the tDNA-PCR-fingerprints of different

Mollicutes species, which is a high number compared to most other bacteria. This is a

somewhat unexpected finding since mollicutes only have a limited number of tRNA genes. In

view of the fact that the tDNA-PCR technique applied on ABI310 only takes into account

small PCR fragments of less than 500 bp, the close proximity of tRNA genes or the possibly

high rate of tDNA-like sequences (14), may partly explain these results. The presence of this

high number of peaks makes the technique well applicable for the identification of a complex

and diverse class of bacteria like the Mollicutes.

In addition, the tDNA-PCR technique has been shown to be very reproducible (4). This is also

apparent from our results, since nearly identical fingerprints were obtained even for strains

that were received from different laboratories and were isolated on different dates. Thanks to

interlaboratory reproducibility (4) and digitised output-data, the tDNA-PCR fingerprints of

more species and subspecies can be collected from different laboratories and published in a

shared online database.

No amplified PCR fragments were observed in 12 cases. The reason for failure is unclear, but

in case of the one M. genitalium and the eight Ureaplasma samples, this is probably due to

poor DNA-quality or possible presence of PCR inhibitors (1), since other strains of the same

species were amplified without problems. For the three M. gallisepticum strains the reason is

less clear since none of the strains yielded a tDNA-PCR pattern despite the high quality of the

used DNA samples as can be concluded from the efficient amplification of the 16S rRNA-

gene. Aligning and comparing all tDNA-sequences of the fully sequenced M. gallisepticum

R-strain with those of other known Mycoplasma tDNA-sequences did not reveal any

exceptional differences (30). Also the arrangement of tDNA-clusters in the M. gallisepticum

genome was very much alike that of other mollicute species (38, 43).


For all other cases, correct identification was obtained, except for the indistinguishable tDNA-

patterns of M. bovis and M. agalactiae. The high similarity of the 16S rDNA sequences of

both species has already been reported (24) and the close relatedness of both species is also

reflected by the fact that M. bovis was first considered as a subspecies of M. agalactiae. Later

studies, involving DNA homology and serology, led to the proposal that M. bovis should not

be regarded as a subspecies of M. agalactiae but as a distinct species (2). In contrast with the

common peaks observed for both species, several minor differences were noted between

individual tDNA-PCR patterns. Important, genetic variability between M. bovis isolates was

already demonstrated using several other molecular DNA techniques (26). These authors

observed two distinct groups of M. bovis isolates and an earlier report also showed the

existence of two distinct groups of M. agalactiae isolates based on antigenic profiles (39).

Whether the presence of the minor peaks coincides with these subgroups is yet unknown.

Still, tDNA-PCR enabled differentiation between subgroups of a number of other species, as

was the case for the strains of A. laidlawii. The two distinct tDNA-PCR profiles may indicate

the existence of two different genomic groups for this species. This has also been indicated by

earlier PFGE results showing different genome sizes for two A. laidlawii strains (32) and

especially by nucleic acid hybridisation studies that demonstrated extensive genomic variation

between different strains (42).

Based on tDNA-PCR, we also observed genotypic diversity within M. genitalium. A recent

report demonstrated the presence of a number of different M. genitalium genotypes (22), but

possible correspondence with the different tDNA-PCR groups established in this study

remains to be studied.

In the obtained dendrogram (Figure 1) some groups, like the M. hominis taxon, clustered

close together similar to phylogenetic data based on 16S rDNA sequence analyses, while

other groups, like the M. neurolyticum taxon, were scattered throughout the dendrogram (31,

47). Therefore, although the use of tDNA-PCR for phylogenetic studies of divergent species

is limited, it can be a helpful tool in resolving taxonomic unclarities for very related species

with almost identical tDNA-PCR patterns. For example, the patterns of the strains belonging

to the species of the mycoides cluster closely resembled each other and were clearly different

from all other species. The finding that the Mycoplasma sp. bovine group 7 strain PG50

(MYP77) showed close resemblance to the M. capricolum strains is in accordance with

suggestions to place these species together in a M. capricolum taxon (8, 9). Our results are

also in agreement with the advice of the subcommittee on the taxonomy of Mollicutes, which


favors the combination of M. mycoides subsp. capri and M. mycoides subsp. mycoides LC

strains into one taxon (8) and a separate position for M. mycoides subsp. mycoides SC.

Accordingly, tDNA-PCR also supports the recent differentiation of Ureaplasma urealyticum

in two distinct species (33). The clustering results indicate the close relationship between

these species, but one of the intergenic tRNA-spacers differs one bp in length between both

species.

The power of the technique was further demonstrated by the fact that tDNA-PCR enabled

detection of mixed cultures. For the mixed cultures of U. urealyticum and M. hominis, the

presence of both species could be recognised because their specific patterns were included in

the constructed consensus library.

In conclusion, tDNA-PCR proved very useful for the identification of mollicute species. The

unexpected high number of peaks provides the technique with a high discriminatory power.

Although tDNA-PCR is especially suited for the identification of unknown isolates, the

technique can be a helpful tool to confirm current and future phylogenetic insights concerning

the subdivision or merging of closely related species. Finally, we could show that tDNA-PCR

can also be used to resolve mixed samples and to point to the existence of additional species.

Acknowledgements This work was supported by a grant of the Belgian Federal Agency of Health, Food Chain

Security and Environment (Grant number S-6136).

The authors kindly thank Dr. Branko Kokotovic (Danish Veterinary Institute, Copenhagen,

Denmark), Dr. Lúcia Manso-Sillivan (CIRAD, Montpellier, France) and Dr. Jozef Bogaerts

(Federal Agency of Health, Food Chain Security and Environment, Brussels, Belgium) for

supplying DNA of M. hyosynoviae isolates, the strains of the mycoides-cluster, and the

M. genitalium samples respectively.

References

1. Al-Soud, W. A., and P. Radstrom. 2001. Purification and characterization of PCR-inhibitory

components in blood cells. J. Clin. Microbiol. 39:485-493.

2. Askaa, G., and H. Erno. 1976. Elevation of Mycoplasma agalactiae subsp. bovis to species rank:

Mycoplasma bovis (Hale et al.) comb. nov. Int. J. Syst. Bacteriol. 26:323-325.


3. Baele, M., P. Baele, M. Vaneechoutte, V. Storms, P. Butaye, L. A. Devriese, G. Verschraegen, M.

Gillis, and F. Haesebrouck. 2000. Application of tRNA intergenic spacer PCR for identification of

Enterococcus species. J. Clin. Microbiol. 38:4201-4207.

4. Baele, M., V. Storms, F. Haesebrouck, L. A. Devriese, M. Gillis, G. Verschraegen, T. de Baere,

and M. Vaneechoutte. 2001. Application and evaluation of the interlaboratory reproducibility of tRNA

intergenic length polymorphism analysis (tDNA-PCR) for identification of Streptococcus species. J.


5. Baele, M., M. Vaneechoutte, R. Verhelst, M. Vancanneyt, L. A. Devriese, and F. Haesebrouck.

2002. Identification of Lactobacillus species using tDNA-PCR. J. Microbiol. Methods. 50:263-271.

6. Ben Abdelmoumen, B., and R. S. Roy. 1995. Antigenic relatedness between seven avian mycoplasma

species as revealed by Western blot analysis. Avian Dis. 39:250-262.

7. Bolske, G. 1988. Survey of Mycoplasma infections in cell cultures and a comparison of detection

methods. Zentralbl. Bakteriol. Mikrobiol. Hyg. 269:331-340.





10. Catry, B., M. Baele, G. Opsomer, A. de Kruif, A. Decostere, and F. Haesebrouck. 2004. tRNA-

intergenic spacer PCR for the identification of Pasteurella and Mannheimia spp. Vet. Microbiol.

98:251-260.

11. De Gheldre, Y., N. Maes, F. L. Presti, J. Etienne, and M. Struelens. 2001. Rapid identification of

clinically relevant Legionella spp. by analysis of transfer DNA intergenic spacer length polymorphism.


12. Edwards, U., T. Rogall, H. Blocker, M. Emde, and E. C. Bottger. 1989. Isolation and direct

complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal

RNA. Nucleic Acids Res. 17:7843-7853.

13. Erno, H., and K. Peterslund. 1983. Growth precipitation test, p. 489-492. In S. Razin, and J. G. Tully

(ed.), Methods in mycoplasmology, vol. I. Academic Press, New York, USA.

14. Frenkel, F. E., M. B. Chaley, E. V. Korotkov, and K. G. Skryabin. 2004. Evolution of tRNA-like

sequences and genome variability. Gene. 335:57-71.

15. Frey, J., X. Cheng, M. P. Monnerat, E. M. Abdo, M. Krawinkler, G. Bolske, and J. Nicolet. 1998.

Genetic and serological analysis of the immunogenic 67-kDa lipoprotein of Mycoplasma sp. bovine

group 7. Res. Microbiol. 149:55-64.




17. Gasparich, G. E., R. F. Whitcomb, D. Dodge, F. E. French, J. Glass, and D. L. Williamson. 2004.

The genus Spiroplasma and its non-helical descendants: phylogenetic classification, correlation with

phenotype and roots of the Mycoplasma mycoides clade. Int. J. Syst. Evol. Microbiol. 54:893-918.

18. Grattard, F., B. Pozzetto, B. de Barbeyrac, H. Renaudin, M. Clerc, O. G. Gaudin, and C. Bebear.

1995. Arbitrarily-primed PCR confirms the differentiation of strains of Ureaplasma urealyticum into

two biovars. Mol. Cell. Probes. 9:383-389.

19. Jensen, J. S., M. B. Borre, and B. Dohn. 2003. Detection of Mycoplasma genitalium by PCR

amplification of the 16S rRNA gene. J. Clin. Microbiol. 41:261-266.

20. Jensen, J. S., S. A. Uldum, J. Sondergard-Andersen, J. Vuust, and K. Lind. 1991. Polymerase chain

reaction for detection of Mycoplasma genitalium in clinical samples. J. Clin. Microbiol. 29:46-50.


22. Ma, L., and D. H. Martin. 2004. Single-Nucleotide Polymorphisms in the rRNA operon and variable

numbers of tandem repeats in the lipoprotein gene among Mycoplasma genitalium strains from clinical

specimens. J. Clin. Microbiol. 42:4876-4878.

23. Maes, N., Y. De Gheldre, R. De Ryck, M. Vaneechoutte, H. Meugnier, J. Etienne, and M. J.

Struelens. 1997. Rapid and accurate identification of Staphylococcus species by tRNA intergenic

spacer length polymorphism analysis. J. Clin. Microbiol. 35:2477-2481.

24. Mattsson, J. G., B. Guss, and K. E. Johansson. 1994. The phylogeny of Mycoplasma bovis as

determined by sequence analysis of the 16S rRNA gene. FEMS Microbiol. Lett. 115:325-328.



Microbiol. 41:4844-4847.

26. McAuliffe, L., B. Kokotovic, R. D. Ayling, and R. A. Nicholas. 2004. Molecular epidemiological

analysis of Mycoplasma bovis isolates from the United Kingdom shows two genetically distinct

clusters. J. Clin. Microbiol. 42:4556-4565.

27. McClelland, M., C. Petersen, and J. Welsh. 1992. Length polymorphisms in tRNA intergenic spacers

detected by using the polymerase chain reaction can distinguish streptococcal strains and species. J.


28. Miles, R., and R. Nicholas. 1998. Mycoplasma protocols, vol. 104. J. M. Walker (ed.) Humana Press,

Totowa, New Jersey, USA.

29. Page, R. D. M. 1996. TREEVIEW: An application to display phylogenetic trees on personal

computers. CABIOS. 12:357-358.

30. Papazisi, L., T. S. Gorton, G. Kutish, P. F. Markham, G. F. Browning, D. K. Nguyen, S. Swartzell,

A. Madan, G. Mahairas, and S. J. Geary. 2003. The complete genome sequence of the avian

pathogen Mycoplasma gallisepticum strain R(low). Microbiology. 149:2307-2316.


31. Pettersson, B., J. G. Tully, G. Bolske, and K. E. Johansson. 2000. Updated phylogenetic description

of the Mycoplasma hominis cluster (Weisburg et al. 1989) based on 16S rDNA sequences. Int. J. Syst.

Evol. Microbiol. 50 Pt 1:291-301.

32. Robertson, J. A., L. E. Pyle, G. W. Stemke, and L. R. Finch. 1990. Human ureaplasmas show

diverse genome sizes by pulsed-field electrophoresis. Nucleic Acids Res. 18:1451-1455.

33. Robertson, J. A., G. W. Stemke, J. W. Davis Jr., R. Harasawa, D. Thirkell, F. Kong, M. C.

Shepard, and D. K. Ford. 2002. Proposal of Ureaplasma parvum sp. nov. and emended description of

Ureaplasma urealyticum (Shepard et al. 1974) Robertson et al. 2001. Int. J. Syst. Evol. Microbiol.

52:587-597.

34. Rodriguez, F., A. Fernandez, and H. J. Ball. 1997. Detection of Mycoplasma mycoides subspecies

mycoides by growth-inhibition using monoclonal antibodies. Res. Vet. Sci. 63:91-92.

35. Salih, B. A., and R. F. Rosenbusch. 2001. Cross-reactive proteins among eight bovine mycoplasmas

detected by monoclonal antibodies. Comp. Immunol. Microbiol. Infect. Dis. 24:103-111.

36. Senterfilt, L. B. 1983. Preparation of antigens and antisera, p. 401-404. In S. Razin, and J. G. Tully

(ed.), Methods in Mycoplasmology, vol. I. Academic Press, New York, USA.

37. Shepard, C. M., and C. D. Lunceford. 1976. Differential agar medium (A7) for identification of

Ureaplasma urealyticum (human T mycoplasmas) in primary cultures of clinical material. J. Clin.


38. Simoneau, P., C. M. Li, S. Loechel, R. Wenzel, R. Herrmann, and P. C. Hu. 1993. Codon reading

scheme in Mycoplasma pneumoniae revealed by the analysis of the complete set of tRNA genes.

Nucleic Acids Res. 21:4967-4974.

39. Solsona, M., M. Lambert, and F. Poumarat. 1996. Genomic, protein homogeneity and antigenic

variability of Mycoplasma agalactiae. Vet. Microbiol. 50:45-58.

40. Stakenborg, T., J. Vicca, P. Butaye, H. Imberechts, J. Peeters, A. de Kruif, F. Haesebrouck, and

D. Maes. 2004. A multiplex PCR to identify porcine mycoplasmas present in broth cultures. Vet. Res.

Comm. In press.

41. Stemke, G. W., F. Laigret, O. Grau, and J. M. Bove. 1992. Phylogenetic relationships of three

porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma

hyorhinis, and complete 16S rRNA sequence of M. flocculare. Int. J. Syst. Bacteriol. 42:220-225.

42. Stephens, E. B., G. S. Aulakh, D. L. Rose, J. G. Tully, and M. F. Barile. 1983. Intraspecies genetic

relatedness among strains of Acholeplasma laidlawii and of Acholeplasma axanthum by nucleic acid

hybridization. J. Gen. Microbiol. 129:1929-1934.

43. Tanaka, R., Y. Andachi, and A. Muto. 1991. Evolution of tRNAs and tRNA genes in Acholeplasma

laidlawii. Nucleic Acids Res. 19:6787-6792.


44. Tully, J. G., and D. L. Rose. 1983. Sterility and quality control of Mycoplasma culture media, p. 121-

135. In S. Razin, and J. G. Tully (ed.), Methods in mycoplasmology: vol. I. Academic Press, New York,

USA.

45. Vaneechoutte, M., P. Boerlin, H. V. Tichy, E. Bannerman, B. Jager, and J. Bille. 1998. Comparison

of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for

atypical Listeria isolates. Int. J. Syst. Bacteriol. 48:127-139.

46. Vaneechoutte, M., G. Claeys, S. Steyaert, T. De Baere, R. Peleman, and G. Verschraegen. 2000.

Isolation of Moraxella canis from an ulcerated metastatic lymph node. J. Clin. Microbiol. 38:3870-

3871.

47. Weisburg, W. G., J. G. Tully, D. L. Rose, J. P. Petzel, H. Oyaizu, D. Yang, L. Mandelco, J.

Sechrest, T. G. Lawrence, J. Van Etten, J. Maniloff, and C.R. Woese. 1989. A phylogenetic

analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171:6455-6467.

48. Welsh, J., and M. McClelland. 1991. Genomic fingerprints produced by PCR with consensus tRNA

gene primers. Nucleic Acids Res. 19:861-866.

A multiplex PCR to identify porcine mycoplasmas present in broth cultures 123

III.3 A MULTIPLEX PCR TO IDENTIFY PORCINE MYCOPLASMAS

PRESENT IN BROTH CULTURES

Tim Stakenborg1, Jo Vicca2, Patrick Butaye1, Hein Imberechts1, Johan Peeters1, Aart

de Kruif2, Freddy Haesebrouck2, and Dominiek Maes2


Belgium

Veterinary Research Communications: in press

124 A multiplex PCR to identify porcine mycoplasmas present in broth cultures

Abstract Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be

present in the lungs of pigs at the same time. These three mycoplasma species all require

similar growth conditions and can be recovered from clinical samples using the same media.

We developed a multiplex PCR as a helpful tool for a rapid differentiation of these three

species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different

forward primers and one single reverse primer were selected. Each forward primer was

compared to available mycoplasma sequences, proving the primers to be specific. The three

amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and

754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no

cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. The

developed multiplex PCR using the proposed set of primers is the first reported assay that

allows the simultaneous identification of the different Mycoplasma species isolated from the

lungs of pigs.

Introduction M. hyopneumoniae is the primary pathogen involved in enzootic pneumonia and is among the

most prevalent agents associated with the porcine respiratory disease complex. Despite the

enormous economical impact of the disease (23), fundamental research is limited due to

demanding isolation techniques. Epidemiological studies are hampered due to difficulties to

detect M. hyopneumoniae strains in pig herds. Recently, several nested PCR assays have been

developed for a direct detection on clinical or environmental samples, but they are unable to

discriminate between viable and non-viable micro-organisms (8, 28). The large benefit of this

technique is its high sensitivity, but extra care is needed since the risk of contamination is

much higher compared to standard PCR methods. In addition, a number of reports indicate the

presence of inhibitory components that may yield false-negative results when working on

clinical samples instead of purified DNA (22, 34). Also enzyme linked immuno-sorbent

assays for the detection of antibodies have been used (12, 27). Due to delay and differences in

time of seroconversion as well as interference of maternal antibodies in young piglets (25),

serological results must be interpreted with care. M. hyorhinis, on the other hand, may cause

serofibrinous to fibrinopurulent polyserositis and arthritis, but is also frequently isolated from

the respiratory tract of healthy pigs. M. flocculare has not been linked to any disease so far,


but the organism is widely spread in swine as well (21). For these economically less important

porcine Mycoplasma spp., even fewer diagnostic kits are available. Therefore, isolation, albeit

labour intensive and limited in sensitivity, remains the gold standard for the diagnosis of

porcine Mycoplasma infections (13, 14, 16, 17, 19, 21).

M. hyopneumoniae, M. hyorhinis and M. flocculare have been reported to cross-react

serologically (6, 31) and exhibit extended phylogenetic similarities (29). The three species are

able to grow in the same media and can complicate unambiguous diagnosis. A fourth porcine

mycoplasma, namely M. hyosynoviae, is associated with arthritis in domestic pigs, but it has

other nutritive requirements. In contrast to the other three Mycoplasma species, this bacterium

is grown in media enriched with arginine (15). Different techniques, including PCR, have

already been reported to differentiate these porcine Mycoplasma species (4, 5, 7, 10, 11, 24,

30), but no single PCR test has been described that simultaneously distinguishes

M. hyopneumoniae, M. hyorhinis and M. flocculare. The aim of this study was to develop

such a multiplex PCR to identify these Mycoplasma species in broth culture. The use of a

multiplex PCR for a rapid differentiation between these species would therefore be time and

money saving.


Mycoplasma strains and cultivation

The reference strains used in this study were the M. hyopneumoniae J strain (ATCC 25934),

the M. flocculare Ms42 strain (ATCC 27399) and the M. hyorhinis BTS-7 strain (ATCC

17981), all kindly provided by Prof. N. Friis (Danish Institute for Food and Veterinary

Research (DFVF), Copenhagen, Denmark). Purified DNA of the M. hyosynoviae S16

reference strain (ATCC 25591) was kindly provided by Dr. B. Kokotovic (DFVF,

Copenhagen, Denmark). Five M. hyopneumoniae, 5 M. hyorhinis and 5 M. flocculare field

strains, all isolated from the lungs of Belgian pigs, were also included in this study.

Cultivation of these mycoplasmas was performed in similarity to earlier reports (14, 16, 17).

Briefly, basal broth medium was composed of 2500 ml Hank’s balanced salt solution, 1400

ml MilliQ H2O, 15 g Brain Heart Infusion (Difco, USA) and 16 g PPLO Broth (Difco). The

mixture was autoclaved at 121°C for 2 minutes and 180 ml of YCS-2 yeast extract (Sigma,

UK), 800 mg bacitracin (Sigma), 500 mg ampicillin (Sigma) and 10 ml of a sterile 0.6%

phenol red solution were added. Horse serum and pig serum was filter-sterilised and added to


the basal broth medium before use. The final medium contained 80% basal medium, 10%

horse serum and 10% pig serum. The pH was adjusted to 7.35 using HCl. Growing cultures

showed a gradually progressing colour change from red to yellow.

Sample preparation method

Genomic DNA of the reference strains was prepared using a phenol-chloroform extraction as

described earlier (3). Since this method is labour intensive, other methods were tried out on

five field isolates of each of the three species. The isolates were grown before processing to a

point where the broth had changed to an orange to yellow colour. In a first approach, one

microliter of the medium with mycoplasmas was used directly in the multiplex PCR. In a

second approach, 1 ml of the growing cultures was spinned down (2’, 10000 g), and the

pellets were resuspended in 100 µl sterile water. After boiling 5 minutes, the samples were

cooled on ice and spinned down again. One µl of the supernatant was used as a template. In a

third method, the mycoplasmas were spinned down and resuspended as in the second method,

but were then incubated in the presence of 2 U of proteinase K during 2 hours at 37°C. Next,

the proteinase K was inactivated at 65°C during 20 minutes and 1 µl of the mixture was used

as a template during PCR. In a final sample preparation method, 1 ml of the broth cultures

was spinned down and resuspended in 50 µl lysis buffer (0.25% SDS in 0.05 N NaOH). After

5 minutes at 95°C, the mixture was cooled down and diluted with 250 µl sterile water. Again

1 µl of the supernatant was used as a template during PCR.

Selection of primers and multiplex reaction

Three specific forward primers were selected based on the aligned 16S rDNA sequences of

M. hyopneumoniae (Genbank accession number: EO2783), M. flocculare (Genbank accession

number: X63377), and M. hyorhinis (Genbank accession number: M24658). One common

reverse primer was selected in a conserved region of the aligned 16S rRNA genes. Based on

these sequences, the theoretical amplification products are 1000 bp (M. hyopneumoniae), 754

bp (M. flocculare), and 1129 bp (M. hyorhinis) in length. The different primers (listed in

Table 1) were combined in a single multiplex reaction. Thirty cycles (30” 94°C; 15” 54.6°C;

and 1’ 68°C) were run on a GeneAmp 9600 Thermal Cycler (Perkin Elmer, USA) using 2.5 U

recombinant Taq DNA polymerase (Invitrogen, The Netherlands), 1x Taq buffer, 75 nmol

MgCl2, 10 nmol of each dNTP, 8 pmol of each forward and 12 pmol of the reverse primer.

The multiplex reaction was tested on purified DNA of the reference strains. To examine the


simultaneous detection of the Mycoplasma species, a DNA sample mix containing 2 ng

genomic DNA of each species was included as well. Besides working on purified genomic

DNA, the multiplex PCR was validated starting directly from growing cultures using the

DNA template preparation methods described above.

Table 1: Primers used in the multiplex PCR.

Primer name Sequence GC% LengthM HYOP FOR 5’ TTCAAAGGAGCCTTCAAGCTTC 3’ 45.5 22 M FLOC FOR 5’ GGGAAGAAAAAAATTAGGTAGGG 3’ 39.1 23

M HYOR FOR 5’ CGGGATGTAGCAATACATTCAG 3’ 45.5 22

M REV 5’ AGAGGCATGATGATTTGACGTC 3’ 45.5 22

Specificity

The specificity of the primers was determined using the Basic Local Alignment Search Tool

(BLAST V2.8.9 [2,191,424 sequences]; (2)). BLAST searches showed the primers to be

highly specific amongst mycoplasmas. No other mycoplasmal sequence, except for those

under investigation, completely matched with the primers. In comparison to other bacterial

genera, homology was only found between the M HYOR 68 FOR primer and most Borrelia

species.

In addition, to ascertain the absence of cross-reactivity between samples, the separate primer

couples were tested in single PCRs. Twenty-five cycles (30” 94°C; 15” 54°C; and 1’ 72°C)

were run on a GeneAmp 9600 Thermal Cycler (Perkin Elmer) using 2.5 U recombinant Taq

DNA polymerase (Invitrogen), 1x Taq buffer, 75 nmol MgCl2, 10 nmol of each dNTP, and 10

pmol of one of the forward primers as well as the reverse primer. For each primer couple, 10

ng genomic template DNA of M. hyopneumoniae, M. hyorhinis, M. hyosynoviae and

M. flocculare, respectively was tested in separate tubes.

Sensitivity

The concentration of genomic DNA of the different Mycoplasma species was determined by

OD260 measuring. A 10-fold serial dilution of the genomic DNA was made and the different

dilutions were tested for their reaction in the multiplex PCR. The minimal dilution still

positive in the multiplex reaction was further diluted 2-fold. The minimum concentration still

showing a positive result was noted.


Results

Multiplex PCR

The multiplex reaction on purified genomic DNA of the three Mycoplasma species generated

the expected bands, which were clearly distinguishable on a 1% agarose gel (Figure 1, lane 1-

3). When using the DNA mix, all three expected bands were observed (Figure 1, lane 5),

while no band was observed with M. hyosynoviae (Figure 1, lane 4).

Figure 1: Multiplex reaction with DNA of M. hyopneumoniae

(lane 1), M. hyorhinis (lane 2), M. flocculare (lane 3), M.

hyosynoviae (negative control, lane 4), and with mixed DNA of

the first three species (lane 5). The SmartLadder (Eurogentec,

Belgium) was used as size-marker.

2000

1500

1000

800

600

400

1 2 3 4 5


Sample preparation method

The multiplex PCR carried out directly on growing cultures or on the boiled mixture gave a

negative result. The use of proteinase K during sample preparation had a positive effect, since

all reactions resulted in the correct PCR product, although the intensity was often faint and

varied between different samples. A good, clear amplification product was obtained for all 15

tested isolates with the fourth preparation method using the alkaline lysis buffer (data not

shown).

Specificity

The three single PCR reactions provided the expected bands of 1129 bp (M. hyorhinis),

1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare), while non-specific bands were

absent (Figure 2). As expected, no PCR product was generated using genomic DNA of

M. hyosynoviae.

Figure 2: A PCR reaction was carried out with primers M REV and M HYOP

FOR (lanes A), M HYOR FOR (lanes B), and M FLOC FOR (lanes C),

respectively. Purified DNA of M. flocculare Ms42 strain (lanes 1),

M. hyorhinis BTS-7 reference strain (lanes 2), and M. hyopneumoniae J-strain

(lanes 3) was used as DNA template. The SmartLadder (Eurogentec) was used

as size-marker.

1500

1000 800

600

400

A1 A2 A3 B1 B2 B3 C1 C2 C3


Sensitivity

Using purified DNA, as little as 500 fg genomic DNA of M. hyorhinis and 1 pg genomic

DNA of M. hyopneumoniae and M. flocculare could be detected (Figure 3).

Discussion M. hyopneumoniae, M. flocculare and M. hyorhinis are fastidious bacteria and are time-

consuming to isolate from porcine lungs. Because they are able to grow in the same isolation

medium, a fast and easy method to differentiate these strains may be a helpful tool during

diagnosis. Therefore, a multiplex PCR was developed. Three different forward primers were

selected in a species-specific region, while the reverse primer was based on a for

mycoplasmas very conserved region of the 16S rRNA gene. The multiplex PCR may

therefore be extended to other Mycoplasma spp. by choosing an appropriate forward primer.

Growing mycoplasma cultures were treated with alkaline lysis buffer before setting up the

multiplex PCR. The importance of PCR-sample processing, prior to the amplification

reaction, was recently reviewed (26). The supplemented sera present in the isolation medium

may have caused the observed inhibitory effect of the PCR reaction when working directly on

broth culture (1). The presence of these inhibitors may also explain the negative results

obtained after boiling. Indeed, PCR inhibitors resistant to heat treatment were reported before

(34). Apparently, at least some of these inhibitors were proteins, since treatment with

Figure 3: Detection limit of the multiplex PCR for M. hyopneumoniae (A), M. flocculare

(B), and M. hyosynoviae (C) performed with 1 ng (lanes 1), 100 pg (lanes 2), 10 pg (lanes

3), 1 pg (lanes 4), 0.5 pg (lanes 5) and 0.25 pg (lanes 6) of purified DNA. The

SmartLadder (Eurogentec) was used as size-marker and the picture was inverted for clarity

reasons.

1500

1000 800 600

400

A 1 A 2 A 3 A 4 A 5 A 6 B 1 B 2 B 3 B 4 B 5 B 6 C 1 C 2 C 3 C 4 C 5 C 6


proteinase K produced the expected PCR fragments. The noted differences between different

samples, the higher costs as well as the long incubation period needed, makes this approach a

less interesting alternative compared to the proposed method using alkaline lysis buffer.

The specificity of the multiplex PCR was tested using each forward primer together with the

common reverse primer in separate PCRs. Another porcine Mycoplasma species,

M. hyosynoviae, cannot be isolated using the same isolation medium (15), but since it is often

found in lungs and tonsils of pigs (18), it was included in the tests. No cross-reaction was

noted. In addition, the primers showed no match with other mycoplasma sequences during our

BLAST-search. Only the M HYOR FOR primer matched with the 16S rDNA of Borrelia spp.

The presence of Borrelia spp. in lungs of pigs has, as far as we know, not been investigated.

Even if so, their growth would be inhibited by the antibiotics present in the used media (20).

Sensitivity testing proved the multiplex reaction to be very sensitive. Since only one copy of

16S rDNA is present in M. hyopneumoniae and M. flocculare (32) and given that the genomic

size of one mycoplasma cell is approximately 1000 kilo base pairs, theoretically as little as

1000 micro-organisms can be detected (given that 1 kbp weighs ~10-3 fg). This is close to the

sensitivity of a PCR reaction of M. hyopneumoniae described by Blanchard et al. (5). A much

higher sensitivity, even on clinical samples, was attained by the use of nested PCR on 16S

sequences of M. hyopneumoniae (9, 28). Since our multiplex PCR is also based on 16S rDNA

sequences, a similar detection limit might be expected using an extra amplification step.

However, since we suggest a first isolation enrichment of the mycoplasmas, sensitivity is of

much less concern.

The multiplex PCR generated species-specific amplicons that were easily distinguishable

using standard gel electrophoresis. Because it is generally accepted that the more efficiently

amplified loci negatively influence the yield of others, only one PCR product is expected to

be visible if one species is strongly dominating (33). Nevertheless, simultaneous detection of

similar amounts of different mycoplasmas was clearly shown.

In conclusion, the multiplex PCR can be used to detect and identify M. hyopneumoniae,

M. flocculare and M. hyorhinis. To our knowledge, this is the first report to simultaneously

differentiate the three Mycoplasma species, potentially present in the lungs of pigs, by means

of a multiplex PCR.


Acknowledgements This work was supported by a grant of the federal agency of Health, Food Chain Security and

Environment (Grant number S-6136). We thank Sara Tistaert for skilful technical assistance.

References

1. Al-Soud, W. A., and P. Radstrom. 2001. Purification and characterization of PCR-inhibitory

components in blood cells. J. Clin. Microbiol. 39:485-493.

2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment

search tool. J. Mol. Biol. 215:403-410.

3. Bashiruddin, J. B. 1998. Extraction of DNA from mycoplasmas. Methods Mol. Biol. 104:141-144.

4. Baumeister, A. K., M. Runge, M. Ganter, A. A. Feenstra, F. Delbeck, and H. Kirchhoff. 1998.

Detection of Mycoplasma hyopneumoniae in bronchoalveolar lavage fluids of pigs by PCR. J. Clin.

Microbiol. 36:1984-1988.

5. Blanchard, B., M. Kobisch, J. M. Bove, and C. Saillard. 1996. Polymerase chain reaction for

Mycoplasma hyopneumoniae detection in tracheobronchiolar washings from pigs. Mol. Cell. Probes.

10:15-22.

6. Bölske, G., M. Strandberg, K. Bergström, and K. Johansson. 1987. Species-specific antigens of

Mycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas. Curr. Microbiol.

15:233-239.

7. Boye, M., T. K. Jensen, P. Ahrens, T. Hagedorn-Olsen, and N. F. Friis. 2001. In situ hybridisation

for identification and differentiation of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae and

Mycoplasma hyorhinis in formalin-fixed porcine tissue sections. APMIS. 109:656-664.

8. Calsamiglia, M., C. Pijoan, and G. J. Bosch. 1999. Profiling Mycoplasma hyopneumoniae in farms

using serology and a nested PCR technique. Swine Health Prod. 7:263-268.

9. Calsamiglia, M., C. Pijoan, and A. Trigo. 1999. Application of a nested polymerase chain reaction

assay to detect Mycoplasma hyopneumoniae from nasal swabs. J. Vet. Diagn. Invest. 11:246-251.

10. Caron, J., M. Ouardani, and S. Dea. 2000. Diagnosis and differentiation of Mycoplasma

hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR and amplification of the p36 and

p46 genes. J. Clin. Microbiol. 38:1390-1396.

11. Dussurget, O., and D. Roulland-Dussoix. 1994. Rapid, sensitive PCR-based detection of

mycoplasmas in simulated samples of animal sera. Appl. Environ. Microbiol. 60:953-959.

12. Feld, N. C., P. Qvist, P. Ahrens, N. F. Friis, and A. Meyling. 1992. A monoclonal blocking ELISA

detecting serum antibodies to Mycoplasma hyopneumoniae. Vet. Microbiol. 30:35-46.

13. Friis, N. 1971. A selective medium for Mycoplasma suipneumoniae. Acta Vet. Scand. 12:454-456.


14. Friis, N. F. 1971. Mycoplasmas cultivated from the respiratory tract of Danish pigs. Acta Vet. Scand.

12:69-79.

15. Friis, N. F. 1974. PhD thesis. Royal Veterinary and Agricultural University.

16. Friis, N. F. 1979. Selective isolation of slowly growing acidifying mycoplasmas from swine and cattle.

Acta Vet. Scand. 20:607-609.

17. Friis, N. F. 1975. Some recommendations concerning primary isolation of Mycoplasma suipneumoniae

and Mycoplasma flocculare a survey. Nord. Vet. Med. 27:337-339.



19. Goodwin, R. F. 1972. Isolation of Mycoplasma suipneumoniae from the nasal cavities and lungs of

pigs affected with enzootic pneumonia or exposed to this infecion. Res. Vet. Sci. 13:262-267.

20. Johnson, S. E., G. C. Klein, G. P. Schmid, and J. C. Feeley. 1984. Susceptibility of the Lyme disease

spirochete to seven antimicrobial agents. Yale J. Biol. Med. 57:549-553.


22. Lantz, P. G., W. Abu al-Soud, R. Knutsson, B. Hahn-Hagerdal, and P. Radstrom. 2000.

Biotechnical use of polymerase chain reaction for microbiological analysis of biological samples.

Biotechnol. Annu. Rev. 5:87-130.

23. Maes, D., H. Deluyker, M. Verdonck, F. Castryck, C. Miry, B. Vrijens, W. Verbeke, J. Viaene,

and A. de Kruif. 1999. Effect of vaccination against Mycoplasma hyopneumoniae in pig herds with an

all-in/all-out production system. Vaccine. 17:1024-1034.

24. Mattsson, J. G., K. Bergstrom, P. Wallgren, and K. E. Johansson. 1995. Detection of Mycoplasma

hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rRNA gene. J. Clin.


25. Morris, C. R., I. A. Gardner, S. K. Hietala, T. E. Carpenter, R. J. Anderson, and K. M. Parker.

1994. Persistence of passively acquired antibodies to Mycoplasma hyopneumoniae in a swine herd.

Prev. Vet. Med. 21:29-41.



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

Jensen, and M. W. Pedersen. 1996. Mycoplasma hyopneumoniae infection in pigs: Duration of the

disease and evaluation of four diagnostic assays. Veterinary Microbiology. 54:23-34.

28. Stärk, K. D., J. Nicolet, and J. Frey. 1998. Detection of Mycoplasma hyopneumoniae by air sampling

with a nested PCR assay. Appl. Environ. Microbiol. 64:543-548.





30. Stemke, G. W., R. Phan, T. F. Young, and R. F. Ross. 1994. Differentiation of Mycoplasma

hyopneumoniae, M. flocculare, and M. hyorhinis on the basis of amplification of a 16S rRNA gene

sequence. Am. J. Vet. Res. 55:81-84.

31. Strasser, M., P. Abiven, M. Kobisch, and J. Nicolet. 1992. Immunological and pathological reactions

in piglets experimentally infected with Mycoplasma hyopneumoniae and/or Mycoplasma flocculare.

Vet. Immunol. Immunopathol. 31:141-153.

32. Taschke, C., M. Q. Klinkert, J. Wolters, and R. Herrmann. 1986. Organization of the ribosomal

RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S

rRNA genes. Mol. Gen. Genet. 205:428-433.

33. Walsh, P. S., H. A. Erlich, and R. Higuchi. 1992. Preferential PCR amplification of alleles:

mechanisms and solutions. PCR Methods Appl. 1:241-250.

34. Wiedbrauk, D. L., J. C. Werner, and A. M. Drevon. 1995. Inhibition of PCR by aqueous and

vitreous fluids. J. Clin. Microbiol. 33:2643-2646.

Diversity of M. hyopneumoniae within and between herds using PFGE 135

III.4 DIVERSITY OF MYCOPLASMA HYOPNEUMONIAE WITHIN

AND BETWEEN HERDS USING PULSED-FIELD GEL ELECTROPHORESIS

Tim Stakenborg1, Jo Vicca2, Patrick Butaye1, Dominiek Maes2, Johan Peeters1, Aart de Kruif2

and Freddy Haesebrouck2


Belgium

Veterinary Microbiology (2005) 109(1-2):29-36.

136 Diversity of M. hyopneumoniae within and between herds using PFGE

Abstract Over the years, pulsed-field gel electrophoresis (PFGE) has been proven a robust technique to

type isolates with a high resolution and a good reproducibility. In this study, a PFGE protocol

is described for the typing of M. hyopneumoniae isolates. The potential of this technique was

demonstrated by comparing M. hyopneumoniae isolates obtained from the same as well as

from different herds. The use of two different restriction enzymes, SalI and ApaI, was

evaluated. For each enzyme, the resulting restriction profiles were clustered using the

unweighted pair group method with arithmetic means (UPGMA). For both obtained

dendrograms, the included isolates of the related M. flocculare species clustered separately

from all M. hyopneumoniae isolates, forming the root of the dendrograms. The PFGE patterns

of the M. hyopneumoniae isolates of different herds were highly diverse and clustered

differently in both dendrograms, illustrated by a Pearson’s correlation coefficient of only 0.33.

A much higher similarity was observed with isolates originating from different pigs of a same

herd. The PFGE patterns of these isolates always clustered according to their herd and this for

both dendrograms. In conclusion, the results indicate a closer relationship of M.

hyopneumoniae isolates within a herd compared to isolates from different herds and this for

both restriction enzymes used. Since the described PFGE technique was shown to be highly

discriminative and reproducible, it will be a helpful tool to further elucidate the epidemiology

of M. hyopneumoniae.

Introduction Respiratory diseases are of major concern for pig herds all over the world. Typically,

M. hyopneumoniae plays an essential role and makes the host more vulnerable to infections

with secondary pathogens (6). Depending on the herd, the symptoms may remain subclinical

or steer towards a severe porcine respiratory disease complex. Herd management and housing

conditions are crucial (19), but also the virulence of the isolate is not to be neglected (38).

Apart from virulence, differences between M. hyopneumoniae isolates were already

demonstrated at antigenic level (28). At least in part, these differences are the result of an

isolate-specific post-translational cleavage, as was shown for the P97 adhesin (5). Also at

genomic level, M. hyopneumoniae isolates turn out to be very heterogeneous. A remarkably

high variety of isolates was observed using AFLP (16), RAPD (1), field inversion gel

electrophoresis (7) or sequence analysis of single genes (39). Further information on the


typing of M. hyopneumoniae is very sparsely available and epidemiological data on the

spreading of the disease are mainly obtained by clinical observations, the detection of serum

antibodies or demonstration of the organism by nested PCR on nasal swabs (9, 10, 37). Direct

contact with infected animals was shown to be a major risk factor (22), but also transmission

by air from other herds or transport vehicles can (re)infect herds originally free of

M. hyopneumoniae (9, 10, 27). Despite these studies, the routes of infection are not always

clear (18) and the spreading of individual clones has not been examined in detail owing to the

limited number of isolates available and the difficulty to standardise currently described

molecular typing techniques. RAPD generally lacks interlaboratory reproducibility (26), while

the high number of fragments generated during AFLP, usually of different intensities,

complicates data processing (11). Multi-locus sequence typing was shown to be a highly

discriminative and reproducible technique, but has not been described for M. hyopneumoniae

and is still too expensive for small or medium-sized laboratories (25). Therefore, pulsed-field

gel electrophoresis (PFGE), also with high discriminatory power and interlaboratory

reproducibility, remains a method of choice for the typing of many bacteria (35, 36).

Although most commonly used to monitor outbreaks, PFGE also allows to examine chronic

infections in order to better understand transmission patterns (33). Therefore, in this study, a

PFGE protocol was optimised and used to compare M. hyopneumoniae isolates obtained

within a herd as well as from different herds.

Materials & Methods

Strains and growth conditions

The J-reference strain (National Collection of Type Cultures (NCTC) 10110), the USA 232

reference strain (21), two Danish field isolates and a total of 35 M. hyopneumoniae isolates,

originating from 21 different Belgian and two different Lithuanian herds, were used (Figures

1 and 2). For both Lithuanian and for 8 Belgian herds, isolates from 2 or 3 different pigs

within the same herd were included. The isolates are indicated using the following format:

‘F1.2A’, where F1 represents the number of the herd, 2 indicates the number of the pig and A

is an arbitrary letter representing the strain. Isolates originating from Lithuania received the

prefix LH, the Danish isolates the prefix DK.

The 232 reference strain was received from the College of Veterinary Medicine (Iowa State

University, USA), while the Danish strains were kindly provided by the Danish Veterinary


Institute (Copenhagen, Denmark). All Belgian and Lithuanian field strains were isolated from

lungs of pigs at slaughter with typical M. hyopneumoniae lesions and positive during

immunofluorescence (15). The isolation was performed in broth medium according to Friis

(8) and the identity of the isolates was confirmed by means of a multiplex-PCR (31). For

PFGE analysis, the isolates were cultivated in 40 ml Friis’ medium (14) at 37°C for at least

five days to the end of the exponential or beginning of the stationary growth phase. The

virulence of 8 isolates has been determined in experimentally inoculated pigs. Isolates F7.2C

and DK Mp143 were of high virulence, isolate F12.6A was moderately virulent and isolates

F1.12A, F5.6A, F9.8K, the J-strain and F13.7B were of low virulence (38). The M. flocculare

Ms42 reference strain (NCTC 10143) and five Belgian M. flocculare field isolates were also

included and served as an outgroup during clustering. The Salmonella enterica serovar

Braenderup reference strain H9812 was used as a size marker as proposed by PulseNet (13,

34) and was grown overnight at 37°C on Columbia agar with 5% ovine blood (Oxoid, UK).

PFGE

The isolates were harvested by centrifugation at 3000 x g for 15 minutes. The supernatant was

placed in a new sterile Falcon tube (BD Biosciences, NJ, USA) and centrifuged a second time

using the same conditions. Both pellets were pooled in 2 ml washing buffer (50 mM Tris-HCl,

10 mM EDTA, 25% (w/v) glucose; pH 7.3) and centrifuged at 13000 x g for 5 minutes. The

washed pellets were resuspended in 800 µl resuspension buffer (75 mM NaCl, 25 mM EDTA;

pH 7.3) and the optical density at 610 nm (OD610) was determined. The bacterial suspension

was adjusted to an OD610 of 1.8 and 200 µl of this suspension was mixed with an equal

volume of 1% Seakem Gold agar (Cambrex Bio Science, Me, USA) at 56°C and poured into

Plexiglas molds (Bio-Rad, Ca, USA) to set into blocks (5x2x10 mm). The blocks were

hardened at 4°C during 10 minutes followed by lysis of the mycoplasma cells using 2 ml

freshly prepared lysis buffer (50 mM EDTA, 1% N-lauroyl-sarcoside, 0.1 mg/ml proteïnase

K, 10 mM Tris-HCl; pH 8.0) for 18 hours at 50°C. Afterwards, the agarose blocks were

washed three times during 15 minutes with distilled water, followed by three washing steps

using sterile washing buffer (50 mM Tris-HCl, 10 mM EDTA; pH 7.3). Next, plugs were

equilibrated during 15 minutes in 1x restriction buffer (delivered with the enzyme).

Subsequently, the DNA in the plugs was digested using restriction buffer containing 30 units

of ApaI (Roche, Switzerland) or SalI (MBI Fermentas, Lithuania) during 4 hours at 37 °C.

Before electrophoresis, the plugs were rinsed with Tris-Borate-EDTA (TBE 0.5x; 45 mM


Tris-borate, 1 mM EDTA, pH 8.0) and loaded in a 1% Seakem Gold agarose (Cambrex Bio

Science). Electrophoresis was performed for 18h under a constant temperature of 14 °C at

6 V/cm and with a linear switch time rampage from 0.5s to 8.5s (CHEF Mapper, Bio-Rad).

Salmonella Braenderup plugs were prepared by the same protocol, but were restricted with

XbaI (Roche). Agarose gels were stained with ethidium bromide and after destaining in water

for 30 minutes, the DNA fragments were visualised using a Genegenius gel documentation

system (Westburg, The Netherlands).

Data analysis and clustering

The digital images were imported in the Bionumerics software (V3.5, Applied Maths,

Belgium) and bands were marked after standardisation using the Salmonella Braenderup

restriction fragments. Calculation of similarity coefficients was performed using the Dice

algorithm. The unweighted pair group method with arithmetic mean (UPGMA) was used for

clustering. In order to attain a complete match between strains analysed in duplicate, the band

position tolerance and optimisation were set to 0.8% and bands smaller than 18 kbp were

omitted. The observed PFGE patterns of strain 232 were compared with the fragments

determined in silico based on its genome sequence (21).

The Pearson’s correlation coefficient was calculated by comparing the Dice similarity

coefficient matrices of both restriction enzymes. In addition to the dendrograms obtained for

both restriction enzymes separately, a cluster analysis on the average of both separate

dendrograms was calculated using BioNumerics, giving both independent analyses the same

equal weight.

The typeability of the PFGE technique for both restriction enzymes was determined. To

calculate the discriminatory power, the Simpson’s index was used (12) with and without

including multiple M. hyopneumoniae isolates originating from a single farm.

Results The described PFGE protocol resulted in clear restriction fragments ranging from 18 to 250

kbp (SalI) or 300 kbp (ApaI). Nicely separated bands were obtained for the

M. hyopneumoniae and the M. flocculare isolates, for both restriction enzymes used (Figure 1

and Figure 2). The Salmonella Braenderup strain suited perfectly as a marker since well

separated bands, spanning the entire size-range, were obvious after restriction with XbaI using


the same protocol (data not shown). For one M. hyopneumoniae isolate, F20.1G, no profile

could be obtained after restriction with ApaI, resulting in a typeability of 97%, compared to

100% for restriction with SalI. A clear, apparent band was visible on top of the gel (data not

shown), representing the unrestricted genomic DNA. All other restriction patterns were

clustered for each restriction enzyme using the UPGMA algorithm.

For strain 232 most restriction fragments determined in silico were observed on gel as well,

although three fragments differed in size. After restriction with ApaI, the calculated band of

190 kbp appeared larger on gel, while the calculated band of 171 kbp was considerably

smaller. After restriction with SalI, the in silico determined band of 90 kbp was only about

half its size on gel.

The Simpson’s index of diversity gave for both restriction enzymes a discrimination index of

0.997 provided that isolates from the same farm were considered related and were not taken

into account. When all M. hyopneumoniae isolates were included, the discrimination index

was still as high as 0.990 for SalI and 0.983 for ApaI.

All M. flocculare isolates included in this study clustered together, separately from the

M. hyopneumoniae isolates (lower than 40% similarity). Only when six or more isolates were

used, the M. flocculare PFGE patterns formed the root of the tree (see Figure 1 and 2).

Whenever less M. flocculare PFGE patterns were used, they clustered together, but in-

between the M. hyopneumoniae isolates (data not shown).

A high variety between the PFGE patterns of M. hyopneumoniae isolates, originating from

different herds, was observed for both restriction enzymes used. Only the isolates from herd

21 and 23 showed identical profiles. With the exception of these latter two isolates and

F18.2A and F4.2C after restriction with ApaI, isolates derived from different farms showed

less than 80% similarity. Clustering of the highly diverse PFGE patterns generated largely

different dendrograms for both restriction enzymes used. In other words, isolates showing a

high similarity based on ApaI results, may differ largely for the SalI restriction patterns, and

vice versa. A weak, but still positive, association between the similarity coefficients for SalI

and ApaI was calculated (Pearson’s correlation coefficient = 0.33).


Figure 1: PFGE patterns of chromosomal DNA of M. hyopneumoniae and M. flocculare

isolates restricted with ApaI. Cluster analysis was performed with UPGMA using the Dice

coefficient and a tolerance and optimisation level of 0.8%. Bands below 18 kbp were omitted

for analysis.


Figure 2: PFGE patterns of chromosomal DNA of M. hyopneumoniae and M. flocculare

isolates restricted with SalI. Cluster analysis was performed with UPGMA using the Dice

coefficient and a tolerance and optimisation level of 0.8%. Bands below 18 kbp were omitted

for analysis.


For the nine isolates tested in an experimental infection model, no linkage between virulence

and PFGE patterns was observed. Neither did isolates of the same geographical origin cluster

together.

Conversely, PFGE patterns of isolates that were obtained from different pigs originating from

the same herd clustered together. This was the case for all isolates analyzed and was apparent

in both dendrograms. With the exception of isolates of herd 19 for restriction with SalI and

Lithuanian herd 3 after restriction with ApaI, isolates derived from the same farm showed

over 80% similarity. The isolates of herd 15, herd 17 and Lithuanian herd 1 had identical

PFGE profiles for both enzymes used. Isolates of herd 11, 14 and 16 on the other hand had

identical profiles for one restriction enzyme, but small differences were observed using the

second restriction enzyme. The multiple isolates of the other herds showed small differences

in their PFGE profiles for both enzymes used.

Discussion The validity of PFGE for molecular typing is well established (20, 33) and its high

discriminatory power and reproducibility was also apparent in this study. Moreover, the

PFGE protocol optimised for M. hyopneumoniae was shown useful for the typing of

M. flocculare isolates as well. This was to be expected since both species are highly related

for both their biochemical and serological characteristics (14, 32). Another porcine

mycoplasma, M. hyorhinis, is less related and initial tests showed indeed that the PFGE

protocol using SalI or ApaI was not useful for the latter species (data not shown).

An enormous heterogeneity between the studied M. hyopneumoniae isolates originating from

different herds was observed. These findings are in agreement with earlier findings obtained

by RAPD (1) and AFLP (16). On the other hand, PFGE patterns of M. hyopneumoniae

isolates originating from a single herd showed more similarity compared to isolates from

different herds. Many strains from the same herd showed even identical PFGE patterns, while

for the other isolates small differences were observed. These differences, together with the

high heterogeneity of strains in general, indicate significant genome plasticity. This is further

substantiated by the comparison of PFGE results and in silico generated data for strain 232.

These observed differences can be explained by a single chromosomal inversion event (from

93-96 kbp to 357-361 kbp). Also for the J reference-strain, genomic differences have been

reported after in vitro passages (7). On the other hand, the similarity of PFGE patterns of

strains originating from a single farm does not automatically imply that these isolates are


related, since PFGE is not suited to depict phylogenetic trees (4, 33, 35). A recent report on

PFGE of Escherichia coli isolates concluded that in the absence of other data, six or more

restriction patterns may be needed to estimate the relatedness of isolates (4). This is in

agreement with our calculation of the Pearson’s correlation coefficient, which was indeed

very low. However, both enzymes show the same trend, namely isolates from the same herd

cluster together. Also, combining the two enzymes in one single cluster-analysis, showed

similar results (data not shown). Although all isolates were obtained from slaughter pigs and

no relation to the age of the pig can be made, these results strongly suggest that isolates from

a single herd are derived from only one or a few ancestral clones.

It is still not known whether these observed genomic differences are linked to phenotypical

differences. Many reports already demonstrated isolate-dependent antigenic variations in

Mycoplasma species (2, 24, 29, 30) and also for M. hyopneumoniae, differences in surface

antigens (40) and lipid content (3) have been reported. If our PFGE results are indeed linked

to differences on the proteonomic level, these data may explain why vaccination, although

normally beneficial, often leads to an incomplete protection that may vary between different

herds (17, 23).

Although PFGE patterns were similar within a herd, an enormous variety between isolates

was even visible for isolates originating from a limited geographical region. These

observations are in contrast with an earlier report where five Swiss strains seemed more

homogenous than five from other origins (7). Probably, more clones of different countries

need to be investigated before definite conclusions can be made. The same report suggested a

possible link between field inversion gel electrophoresis patterns and virulence (7), but again

this could not be confirmed with our data.

In conclusion, the PFGE profiles of M. hyopneumoniae isolates originating from different

herds were very diverse, compared to the limited heterogeneity seen within a herd. Further

research may be needed to sustain these data and to further elucidate the distribution, stability

and persistence of M. hyopneumoniae clones. The proposed PFGE protocol proved a very

useful and reproducible tool to perform these studies.


Acknowledgements This work was supported by a grant of the Federal Service of Public Health, Food Chain


The authors thank Véronique Collet and Sara Tistaert for skilful technical assistance and

Annelies Pil for the numerous inspiring discussions.

References



2. Bhugra, B., L. L. Voelker, N. Zou, H. Yu, and K. Dybvig. 1995. Mechanism of antigenic variation in

Mycoplasma pulmonis: interwoven, site-specific DNA inversions. Mol. Microbiol. 18:703-714.

3. Chen, J. W., L. Zhang, J. Song, F. Hwang, Q. Dong, J. Liui, and Y. Qian. 1992. Comparative

analysis of glycoprotein and glycolipid composition of virulent and avirulent strain membranes of

Mycoplasma hyopneumoniae. Curr. Microbiol. 24:189-192.

4. Davis, M. A., D. D. Hancock, T. E. Besser, and D. R. Call. 2003. Evaluation of pulsed-field gel

electrophoresis as a tool for determining the degree of genetic relatedness between strains of

Escherichia coli O157:H7. J. Clin. Microbiol. 41:1843-1849.


processing of the Mycoplasma hyopneumoniae cilium adhesin. Infect. Immun. 72:2791-2802.

6. Done, S. 2002. How respiratory pathogens damage the pig. Pig International. 32:35-36.

7. Frey, J., A. Haldimann, and J. Nicolet. 1992. Chromosomal heterogeneity of various Mycoplasma

hyopneumoniae field strains. Int. J. Syst. Bacteriol. 42:275-280.



9. Goodwin, R. F. 1985. Apparent reinfection of enzootic-pneumonia-free pig herds: search for possible

causes. Vet. Rec. 116:690-694.




43:145-156.

11. Hong, Y., and A. Chuah. 2003. A format for databasing and comparison of AFLP fingerprint profiles.

BMC Bioinformatics. 4:7.


12. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing

systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26:2465-2466.

13. Hunter, S. B., P. Vauterin, M. A. Lambert-Fair, M. S. Van Duyne, K. Kubota, L. Graves, D.

Wrigley, T. Barrett, and E. Ribot. 2005. Establishment of a universal size standard strain for use with

the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases

to the new size standard. J. Clin. Microbiol. 43:1045-1050.


15. Kobisch, M., J. Tillon, P. Vannier, S. Magneur, and P. Morvan. 1978. Pneumonie enzootique à

Mycoplasma suipneumoniae chez le porc: diagnostic rapide et recherches d'anticorps. Rec. Méd. Vet.

154:847-852.



17. Maes, D., H. Deluyker, M. Verdonck, F. Castryck, C. Miry, B. Vrijens, W. Verbeke, J. Viaene,

and A. de Kruif. 1999. Effect of vaccination against Mycoplasma hyopneumoniae in pig herds with an

all-in/all-out production system. Vaccine. 17:1024-1034.

18. Maes, D., M. Verdonck, and A. de Kruif. 2000. Enzoötische pneumonie bij varkens. Deel I: De

ziekte. Tijdschr. Diergeneeskd. [in Dutch]. 69:94-100.

19. Maes, D., M. Verdonck, H. Deluyker, and A. de Kruif. 1996. Enzootic pneumonia in pigs. Vet Q.

18:104-109.

20. Maule, J. 1998. Pulsed-field gel electrophoresis. Mol. Biotechnol. 9:107-126.




22. Morris, C. R., I. A. Gardner, S. K. Hietala, T. E. Carpenter, R. J. Anderson, and K. M. Parker.

1995. Seroepidemiologic study of natural transmission of Mycoplasma hyopneumoniae in a swine herd.

Prev. Vet. Med. 21:323-337.

23. Morrow, W., G. Iglesias, C. Stanislaw, A. Stephenson, and G. Erickson. 1994. Effect of a

mycoplasma vaccine on average daily gain in swine. Swine Hlth. 2:13-18.

24. Noormohammadi, A. H., P. F. Markham, K. G. Whithear, I. D. Walker, V. A. Gurevich, D. H.

Ley, and G. F. Browning. 1997. Mycoplasma synoviae has two distinct phase-variable major

membrane antigens, one of which is a putative hemagglutinin. Infect. Immun. 65:2542-2547.







27. Rautiainen, E., and P. Wallgren. 2001. Aspects of the transmission of protection against Mycoplasma

hyopneumoniae from sow to offspring. J. Vet. Med. B. Infect. Dis. Vet. Public Health. 48:55-65.

28. Ro, L. H., and R. F. Ross. 1983. Comparison of Mycoplasma hyopneumoniae strains by serologic

methods. Am. J. Vet. Res. 44:2087-2094.

29. Rosengarten, R., P. M. Theiss, D. Yogev, and K. S. Wise. 1993. Antigenic variation in Mycoplasma

hyorhinis: increased repertoire of variable lipoproteins expanding surface diversity and structural

complexity. Infect. Immun. 61:2224-2228.

30. Roske, K., A. Blanchard, I. Chambaud, C. Citti, J. H. Helbig, M. C. Prevost, R. Rosengarten, and

E. Jacobs. 2001. Phase variation among major surface antigens of Mycoplasma penetrans. Infect.

Immun. 69:7642-7651.

31. Stakenborg, T., J. Vicca, P. Butaye, H. Imberechts, J. Peeters, A. de Kruif, F. Haesebrouck, and

D. Maes. 2004. A multiplex PCR to identify porcine mycoplasmas present in broth cultures. Vet. Res.

Comm. in press.




33. Struelens, M. J., R. De Ryck, and A. Deplano. 2001. Analysis of microbial genomic macrorestriction

patterns by Pulsed-Field Gel Electrophoresis (PFGE) typing, p. 159-176. In L. Dijkshoorn, K. J.

Towner, and M. Struelens (ed.), New approaches for the generation and analysis of microbial typing

data. Elsevier, B.V., Amsterdam.



7:382-389.

35. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B.

Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel

electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.

36. van Belkum, A., W. van Leeuwen, M. E. Kaufmann, B. Cookson, F. Forey, J. Etienne, R. Goering,

F. Tenover, C. Steward, F. O'Brien, W. Grubb, P. Tassios, N. Legakis, A. Morvan, N. El Solh, R.

de Ryck, M. Struelens, S. Salmenlinna, J. Vuopio-Varkila, M. Kooistra, A. Talens, W. Witte, and

H. Verbrugh. 1998. Assessment of resolution and intercenter reproducibility of results of genotyping

Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a

multicenter study. J. Clin. Microbiol. 36:1653-1659.


37. Vicca, J., D. Maes, L. Thermote, J. Peeters, F. Haesebrouck, and A. de Kruif. 2002. Patterns of

Mycoplasma hyopneumoniae infections in Belgian farrow-to-finish pig herds with diverging disease-

course. J. Vet. Med. B. Infect. Dis. Vet. Public Health. 49:349-353.

38. Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. Haesebrouck. 2003.

Evaluation of virulence of Mycoplasma hyopneumoniae field isolates. Vet. Microbiol. 97:177-190.



40. Wise, K. S., and M. F. Kim. 1987. Major membrane surface proteins of Mycoplasma hyopneumoniae

selectively modified by covalently bound lipid. J. Bacteriol. 169:5546-5555.

Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 149

III.5 COMPARISON OF MOLECULAR TECHNIQUES FOR THE

TYPING OF MYCOPLASMA HYOPNEUMONIAE ISOLATES

Tim Stakenborg*1, Jo Vicca2, Dominiek Maes2, Johan Peeters1, Aart de Kruif2, Freddy

Haesebrouck2, and Patrick Butaye1


Belgium

150 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates

Abstract In this study, we compared the potential of amplified fragment length polymorphism (AFLP),

random amplified polymorphic DNA (RAPD) analysis, restriction fragment length

polymorphism (RFLP) of the gene encoding lipoprotein P146, and the variable number of

tandem repeats (VNTR) of the P97 encoding gene, as possible methods to type an

international collection of M. hyopneumoniae isolates. All techniques showed a typeability of

100% and high intraspecific diversity. However, the discriminatory power of the different

techniques varied considerably. AFLP (>0.99) and PCR-RFLP of the P146 encoding gene

(>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97

(<0.92). Other, preferentially well spread, tandem repeat regions should be included in order

for this latter technique to become valuable for typing purposes. RAPD was also found a less

interesting typing technique because of its low reproducibility between different runs.

Nevertheless, all molecular techniques showed overall more resemblance between strains

isolated from different pigs from a same herd. On the other hand, none of the techniques was

able to show a clear relationship between the country of origin and the obtained fingerprints.

We conclude that AFLP and an earlier described PFGE technique are highly reliable and

discriminatory typing techniques to outline the genomic diversity of M. hyopneumoniae

isolates. Our data also show that RFLP of a highly variable gene encoding P146 may be an

equally useful alternative to demonstrate intraspecific variability, although the generation of

sequence variability of the gene remains unclear and must be further examined.


Introduction Mycoplasma hyopneumoniae is the primary cause of enzootic pneumonia in pigs. Although

vaccines have been developed, infections are still hard to control (20) and, even in countries

aiming to eradicate enzootic pneumonia, re-infections occur frequently (11). The disease is

not associated with a high mortality rate, but the severity may vary greatly between different

herds (34). Farm management is considered essential (7), but also the intrinsic virulence of

circulating M. hyopneumoniae strains has been proven an important cause for this variation

(34). The underlying mechanism to explain these results has remained elusive, although

several techniques demonstrated M. hyopneumoniae to be a highly heterogeneous species.

Analysis of the proteome showed different SDS-PAGE profiles for different isolates (5) that

were at least partly the result of strain-specific post-translational modifications (6). On

genomic level, an enormous heterogeneity was demonstrated by various typing techniques

such as random amplified polymorphic DNA (RAPD) (1), amplified fragment length

polymorphism (AFLP) (19), or pulsed-field gel electrophoresis (PFGE) (30). Moreover, the

number of a yet unassigned insertion-like sequence varied between different strains (10) and

also differences in the reiterated regions of a P97 adhesin encoding gene were reported for

different isolates (14, 16). All these different techniques may prove useful in future

epidemiological studies to trace strains or to visualise infection patterns. To perform such

epidemiological studies, the choice of the typing technique is essential. However, in case of

M. hyopneumoniae, the value of different typing techniques has never been assessed. As long

as whole genome sequencing is not easily attainable, typing techniques, which ideally

represent the true phylogenetic relation between strains, are bound to their own intrinsic

limitations. In this study, we compared the use of formerly described techniques (RAPD and

AFLP) and newly PCR based techniques (PCR-RFLP of the P146 gene and the VNTR of the

P97 gene) as possible methods to study the diversity of M. hyopneumoniae strains. For each

typing technique, the discriminatory power, reproducibility and ease of performance were

compared using an identical set of strains. The obtained results were discussed in detail and

compared with PFGE data on a similar set of isolates described earlier by our group (30).


Materials & Methods

Bacterial isolates, media, and DNA extraction

A total of 43 M. hyopneumoniae isolates were used together with reference strains J (ATCC

25934), USA 232 (21), and M. flocculare Ms42 (ATCC 27399). All Belgian and Lithuanian

field isolates were derived from lung samples of pigs at slaughter. These isolates received a

name of the format: ‘F1.2A’, where F1 represents the number of the herd, 2 indicates the

number of the pig and A is an arbitrary letter representing the isolate. Isolates from different

pigs from the same herd were obtained from lung samples collected at the same moment. For

strains that were received from other laboratories, the genuine strain designation was kept

unchanged. For clarity reasons, the international code representing the country of origin was

always indicated between parentheses after the isolate’s name. Further information about the

included strains is listed, in as much detail as possible, in Table 1.

Friis’ broth was used to grow both the M. flocculare and M. hyopneumoniae strains (9).

Purified, genomic DNA was prepared using a phenol/chloroform extraction method (2).

RAPD

For RAPD analyses, 45 cycles (1’ 94°C; 1’ 36°C; and 2’ 72°C) were run on a GeneAmp 9600

Thermal Cycler (Perkin Elmer, Ma, USA) using 20 pmol of a primer OPA-3 (5’

AGTCAGCCAC) described by Artiushin and Minion (1), and exactly 30 ng of purified,

genomic DNA as a template. To minimise the variability between different runs, ready-to-go

RAPD-beads (Amersham Biosciences, Germany) were used and all samples were run

simultaneously during one single PCR. After amplification, 10 µl of the PCR mixture was

analyzed by electrophoresis (120V, 90’) on 1% agarose gel (Sigma, UK). The DNA

fragments were visualised using a GeneGenius gel documentation system (Westburg, The

Netherlands) and exported to Bionumerics (V3.5, Applied-Maths, Belgium) for further

analysis. Bands annotated by the software were visually controlled and fragments smaller than

500 bp were omitted for further analysis. Calculation of similarity coefficients was performed

using the Dice algorithm. The unweighted pair group method with arithmetic means

(UPGMA) was used for clustering with a band position tolerance and optimisation setting of

1%.


Table 1: Overview of the M. hyopneumoniae strains used in this study and the estimated number of reiterated repeats of P97.

Estimated number of reiterated repeats Farm

number

Pig Strain designation

Year of isolation Place, country of origin1

Number of

in vitro passages

RR1 RR2

F1 12 A 2000 Nieuwekapelle, Belgium 9 2.0 3.0 F2 3 K 2000 Wuustwezel, Belgium 9 12.0 4.9 F3 1 M 2000 Namen, Belgium 10 20.9 2.9 F4 2 C 2001 Moorsele, Belgium 6 12.9 3.0 F5 6 A 2000 Loenhout, Belgium 19 16.4 3.9 F6 12 D 2000 Linter, Belgium 8 9.1 4.9 F7 2 C 2000 Landegem, Belgium 8 16.5 2.9 F8 3 C 2001 Diksmuide, Belgium 18 13.4 2.9 F8 5 L 2001 Diksmuide, Belgium 9 13.4 2.8 F9 8 K 2001 Diksmuide, Belgium 15 11.1 2.9

F10 7 E 2001 Beveren, Belgium 8 9.1 3.0 F11 1 A 2001 Veurne, Belgium 7 10.6 3.0 F11 8 A 2001 Veurne, Belgium 7 10.6 3.0 F12 6 A 2001 Linter, Belgium 6 10.7 3.0 F13 7 B 2001 Poperinge, Belgium 10 14.0 2.9 F13 10 A 2001 Poperinge, Belgium 10 14.1 2.9 F14 7 E 2001 Minderhout, Belgium 8 12.9 2.9 F14 9 A 2001 Minderhout, Belgium 8 12.9 2.9 F15 2 A 2001 Olen, Belgium 8 8.0 4.0 F15 3 L 2001 Olen, Belgium 15 8.0 3.9 F15 10 A 2001 Olen, Belgium 6 8.0 4.0 F16 2 X 2001 Olen, Belgium 8 8.1 2.9 F16 4 B 2001 Olen, Belgium 6 13.1 2.9 F17 1 J 2002 Sluizen, Belgium 16 13.0 2.9 F17 2 N 2002 Sluizen, Belgium 5 13.0 2.9 F18 2 A 2002 Slijpe, Belgium 6 12.3 3.9 F19 1 E 2002 Leffinge, Belgium 7 11.0 2.9 F19 4 A 2002 Leffinge, Belgium 21 11.1 2.9 F19 6 E 2002 Leffinge, Belgium 6 11.0 2.9 F21 9 C 2002 Bocholt, Belgium 13 10.3 3.9 F23 7 E 2002 Waasmunster, Belgium 9 10.1 3.9

- - J ~1965 (ATCC 27715)2 NA 9.0 4.9 LH1 2 A 2003 Vilnius, Lithuania 6 14.1 2.9 LH1 3 B 2003 Vilnius, Lithuania 8 14.3 2.9 LH3 1 B 2003 Vilnius, Lithuania 16 12.4 2.9 LH3 3 B 2003 Vilnius, Lithuania 16 12.3 2.9

- - MP143 NA Denmark NA 11.1 2.9 - - SVS22 2000 Denmark NA 10.8 2.8 - - Mp18 1998 Denmark NA 11.0 2.9 - - 232 NA USA3 20 14.2 3.9

NL2 6 B NA The Netherlands NA 14.3 5.9 NL3 4 A NA The Netherlands NA 13.5 2.9

- - W79 ~1995 United Kingdom NA 12.4 1.9 - - W58 ~1995 United Kingdom NA 15.7 1.9 - - E62 ~1995 United Kingdom NA 11.5 3.9

1 strains originating from Denmark were kindly provided by Dr. F. Friis (Danish Veterinary Institute, Copenhagen,

Denmark), from the USA by Dr. E. Thacker (Iowa State University, USA), from The Netherlands by Dr. A. van Essen

(Animal Sciences Group, Wageningen University and Research Centre, The Netherlands), and from the UK by Dr. H.

Windsor (Mycoplasma Experience, Surrey, UK). The Lithuania strains were isolated from porcine lungs kindly provided

by Dr. K. Garlaite (Lithuanian Veterinary Academy, Vilnius, Lithuania). 2 NA = not availble 3 strain 232 was isolated originally from a pig infected with M. hyopneumoniae strain 11 (ATCC 27714) (21).


AFLP

AFLP was performed in similarity to an earlier report (19). Briefly, 200 ng genomic DNA

was diluted in 20 µl restriction buffer (SuRE/Cut Buffer M, Roche, Switzerland) and

restricted with 10 U BglII (Roche) and 10 U MfeI (Fermentas, Lithuania) during 3 hours at

37°C. After incubation for 15’ at 65°C, a 20 µl ligation reaction was set up using 5 µl of the

digested DNA, 2 pmol of the BGL-adapter, 20 pmol of the MFE-adapter (19), 1 U T4-ligase

(Amersham), 2 µl restriction buffer (Amersham), and 8 µl restriction buffer (Amersham).

Ligation was carried out overnight at 16°C. The succeeding amplification reaction was

performed as noted in Table 2 using 2 µl of the 10-fold diluted ligation product as template.

One µl of the amplified PCR products were diluted in 40 µl sample loading solution

(Beckman, UK) supplemented with CEQ Size-standard 600 (Beckman) and ran on a

CEQ8000 Genetic Analysis System (Beckman) for separation and visualisation. Obtained raw

data were subsequently exported to Bionumerics (Applied-Maths) and converted to gel

images. After normalisation, fragments between 60 bp and 560 bp were defined. Clustering

analysis of the obtained fingerprints was performed with UPGMA on the basis of a similarity

matrix with calculated Jaccard’s similarity coefficients. For clustering, the tolerance and

optimisation level was set to 0.7%.

To determine the reproducibility of the AFLP procedure, three independent DNA samples of

10 arbitrarily chosen strains were analysed on different days.

PCR-RFLP analysis of the P146 encoding gene

For the amplification of the P146 gene, a PCR was performed using primers and reaction

conditions noted in Table 2. Some strains yielded a faint non-specific PCR fragment of about

900 bp in size and for these, the PCR was repeated in nested format using a pre-amplification

step as noted in Table 2. After PCR, about 100 ng of the final PCR product was digested

during 3 hours at 37°C in restriction buffer (SuRE/Cut Buffer A, Roche) containing 10 U of

restriction enzyme AluI (Roche). Restricted fragments were separated during 2 hours at 120 V

on a 2% Nusieve agar (Cambrex BioScience). The 50 bp O’RangeRuler (Fermentas) was used

as size standard and was loaded at least twice for every 10 samples. After electrophoresis,

DNA fragments were visualised using a GeneGenius gel documentation system (Westburg).

The digital images were exported to Bionumerics (Applied-Maths) for standardisation and

annotation of the bands. Fragments smaller than 175 bp were omitted from the analysis.


Levels of similarity between fingerprints were calculated employing the Dice algorithm. In

order to attain a complete match between strains analysed in duplicate, the tolerance and

optimisation level was both set to 1%. Cluster analysis was performed with UPGMA.

To verify the accuracy of the technique, the in vitro observed restriction patterns of isolate

F7.2C and USA 232 were compared with those calculated in silico based on the P146 gene

sequences (see further). To check whether in vitro cultivation influenced results, the test was

repeated on three strains after 5, 10, and 15 in vitro subcultivation steps.

VNTR present in the P97 encoding gene

Two different reiterated repeat regions (RR1 and RR2) have been described for the P97

adhesin gene of M. hyopneumoniae (14). For each strain, two PCRs were performed to

selectively amplify the RR-regions using the primers and cycle conditions stated in Table 2.

Amplified fragments were separated on a 2% Nusieve agar (Cambrex Bio Science, Me, USA)

during 2 hours at 120V and visualised using a GeneGenius gel documentation system

(Westburg). Based on a 50 bp O’RangeRuler (Fermentas), which was loaded at least twice for

every 10 samples, sizes of amplified fragments were estimated using Kodak digital science

1D software (V3.0, Kodak Company, NY, USA). The accuracy of the technique for

estimation of the number of repeats was examined by sequence analysis of the repeat regions

for 10 arbitrarily chosen isolates (see further). In addition, the standard deviation of the

technique was calculated by comparing the expected size of the amplified RR2 region (i.e.

number of RR2-repeats times 30 bp plus 194 bp) with the size of the amplification products

observed on gel. To check whether strains could be safely grown in the laboratory, the

number of repeats was compared for three arbitrarily chosen strains that were subcultivated 5,

10, and 15 times in vitro.

Discriminatory power

The Simpson’s index of diversity was calculated for each technique (17). Since the

dependency between isolates originating from a single herd was unknown, two different

discriminatory indexes were calculated, one including all M. hyopneumoniae strains and one

excluding all isolates that had an identical fingerprint and originated from a single herd. This

implies that in case some isolates of the same herd represent an identical clone, the true value

of the Simpson’s index of diversity should fall between these two estimates.


Sequence analysis

Sequencing of the gene encoding lipoprotein P146 of strain F7.2C and part of the genes

encoding P97 of 10 arbitrarily chosen isolates (Table 3) was performed on PCR products.

Samples were purified with QIAquick spin columns (Qiagen, Germany) and sequenced on a

CEQ8000 Genetic Analysis System (Beckman, UK) by using the Quickstart kit (Beckman)

according to the manufacturer’s instructions. The obtained sequences were exported to

VectorNTI (V9, Informax, Invitrogen) for assemblage and further analysis. The sequence of

the P146 gene of isolate F7.2C was submitted to Genbank (accession nr. DQ088147).

Table 2: Primers and cycle conditions used in this study. Target sequence Sequence (5’ -> 3’) Number of cycles (cycle conditions)1

Primer

AFLP PCR2 30 ( 1’ 94°C; 1’ 54°C ; and 90” 72°C)

BGL-2F* (D4*)GAGTACACTGTCGATCT

MFE-1 GAGAGCTCTTGGAATTG

P146 (pre-amplification) 30 (15” 94°C; 30” 51°C; and 1’ 72°C)

P146 cFOR CATTAGTAACAGCAACAGCCATTG

P146 cREV TACCTCGCCGCCTTAGCAG

P146 (amplification) 25 (15” 94°C; 30” 52.5°C; and 1’ 72°C)

P146 FOR TTAGTAACAGCAACAGCCATTG

P146 REV CCCTTAAGTGGACAATTTTAGC

P97 (repeat region 1) 30 (30” 94°C; 30” 53.7°C; and 1’ 72°C)

RR1 FOR GAAGCTATCAAAAAAGGGGAAACTA

RR1 REV GGTTTATTTGTAAGTGAAAAGCCAG

P97 (repeat region 2) 30 (1’ 94°C; 1’ 50.3°C; and 45” 72°C)

RR2 FOR AGCGAGTATGAAGAACAAGAA

RR2 REV TTTTTACCTAAGTCAGGAAGG 1 All PCRs, unless stated otherwise (see postscript 2), were performed using 3 U of recombinant Taq polymerase

(Invitrogen), 5 µl of PCR buffer (Invitrogen) including 2 mM MgCl2, 0.2 mM of each dNTP and 10 pmol of both

forward and reverse primer. 2 The AFLP-PCR was performed using 2 U of AmpliTaq polymerase (Amersham Biosciences), 5 µl of PCR buffer

II (Amersham Biosciences) supplemented with 2.5 mM MgCl2, 0.2 mM of each dNTP and 10 pmol of both

forward and reverse primer.


Results

RAPD

Since RAPD patterns between different runs were in our hands not reproducible (data not

shown), even not with the use of an as much as possible standardised method, the analysis of

all samples was carried out during one single run. A limited number of fragments (two to

eight) were observed for each isolate. All isolates showed a band of about 1300 bp in size and

for most M. hyopneumoniae isolates another band of about 550 bp was observed. For the

M. flocculare Ms42 strain, an intense band of about 750 bp was observed, but it could not

been used for species differentiation as fragments of a similar size were observed for some

Danish M. hyopneumoniae field isolates as well (Figure 1). The intensity of many bands

between non-identical patterns varied and complicated analysis.

Isolates originating from the same herd had identical RAPD patterns, with the exception of

isolates from herd F8 (BE), F16 (BE), F19 (BE), and LH3 (LT). On the other hand, also many

strains originating from different herds had identical profiles, resulting in a discriminatory

index of 0.95 (for both calculated indexes).

AFLP

All M. hyopneumoniae isolates generated about 100 clearly separated fragments, with the

exception of the isolates of herd F19 (BE), F21 (BE), and F23 (BE), which showed more

bands, and of the isolate F2.3K (BE), which showed considerably fewer bands. The M.

flocculare Ms42 strain showed a clearly different and less complex pattern and formed the

root of the dendrogram. Reproducibility tests showed similar peak profiles, although peak

intensities often varied. After normalisation and band annotation, all replicates showed

similarity values of at least 92% (data not shown). This value was used a cut-off value to

differentiate between isolates. Despite this cut-off value, only the multiple isolates originating

from herd F17 (BE), F19 (BE), and LH1 (LT) were indistinguishable. Also the AFLP patterns

of F21.9C (BE) and F23.7E (BE) were considered identical. All other isolates had similarity

values below the cut-off value (Figure 2). This corresponded to a discriminatory index

calculated higher than 99% (with and without the inclusion of multiple isolates of the same

herd).


Figure 1: RAPD patterns of the M. hyopneumoniae isolates and the M. flocculare strain

Ms42. Cluster analysis was performed with UPGMA using the Dice coefficient and a

tolerance and optimisation level of 1%. Bands below 500 bp were omitted for analysis.


Figure 2: Dendrogram of the obtained AFLP fragments from 60 to 560 bp in size. Cluster

analysis was performed with UPGMA using the Jaccard’s coefficient and a tolerance and

optimisation level of 0.7%. The dashed line represents the cut-off value (92%) for similarity

determined by analysis of replicates. Patterns with a higher similarity value are considered

indistinguishable. The included M. flocculare strain Ms42 served as an outgroup.


Figure 3: PCR-RFLP patterns of the P146 gene the M. hyopneumoniae isolates. Cluster

analysis was performed with UPGMA using the Dice coefficient and a tolerance and

optimisation level of 1%. Bands below 175 bp were omitted for analysis.


PCR-RFLP analysis of the P146 encoding gene

Restriction analysis with AluI showed an extensive variation in the P146 gene of different

isolates. This variation was further illustrated by the high Simpson’s index of diversity, which

was calculated to be higher than 0.98 without and higher than 0.97 with the inclusion of

isolates originating from the same farm. In contrast to this enormous variation, isolates

originating from the same herd had identical profiles in 6 out of 10 cases (Figure 3). Also the

restriction profiles of three strains that were subcultured up to 15 times in the laboratory were

identical (data not shown).

Restriction patterns calculated in silico for the determined sequence of the P146 encoding

gene of strain F7.2K (BE) and strain USA232 (21) corresponded to those observed on gel. By

comparison of the two DNA sequences, several highly variable repeat regions were observed,

mainly in the C-terminal part of the gene. These regions included a poly-serine chain of

variable length, a repeat region rich in proline and glutamine residues of variable length that

could be represented by the following format [Q]n[(P/S)Q]m, and a variable poly-alanine chain

situated directly before the stop codon of the gene.

VNTR present in the P97 encoding gene

Both the RR1 (15 bp in length) and the RR2 repeat (30 bp in length) have been described in

detail before (14, 35). As shown in Table 1, the estimated number of RR1 repeats ranged for

most strains from 8 to 16 copies. However, two extremes were noted, isolate F1.12 (BE) with

only 2 copies, and isolate F3.1M (BE) with 21 copies of the RR1 repeat. The number of RR2

repeats was less diverse and ranged from 2 to 6 copies, with most isolates having 3 copies.

The number of repeat regions of isolates originating from the same herd was identical, except

for the two isolates of herd F16 (BE) where a difference between the number of RR1-repeats

was noted. This resulted in a discriminatory power as low as 0.90 for RR1 and 0.59 for RR2

when excluding replicates or 0.88 for RR1 and 0.53 for RR2 when including all isolates of the

same herd as well. By combining the two repeats, the discriminatory power raised to 0.91

with and 0.94 without the inclusion of multiple isolates per herd.

The calculated standard deviation on the estimated size was 3.0 bp, while the maximum error

observed between the expected fragment size and the size determined on gel was 7 bp.

Therefore, the technique can be used to exactly determine the number of RR2 repeats. In case

of RR1 repeats, and as verified by sequence analyses, the copy number is merely an estimate

of the true value. Apparently, RR1-repeats are followed by another repeat region of the format


GCT(ACTAAT)nACT, where n represents a number from 1 to 6 (Table 3). Since the region

consists out of repeated threonine and asparagines residues, the repeat is further referred to as

TN-repeat.

The number of repeats did not appear to change easily over in vitro passages, since bands of

identical size were observed for three strains subcultivated 5, 10 and 15 times in vitro (data

not shown).

Table 3: The by PCR estimated number of RR-repeats of the P97 gene of strain USA232 and

10 arbitrarily chosen isolates compared with the actual number of RR-repeats

determined by sequence analysis. RR1 RR2

Strain Estimated length

Actual length

Estimated RR1

repeats1

Actual RR1

repeats

Number TN

repeats2

Estimated length

Actual length

Estimated RR2

repeats3

Actual RR2

repeats F1.12K 184 185 1.9 2 3 284 284 3.0 3 F5.6B 403 401 16.5 16 4 312 314 3.0 3 F6.12D 292 288 9.1 8 5 341 344 3.9 4 F7.2C 399 397 16.3 15 6 282 284 4.9 5 F9.8K 322 320 11.1 11 3 280 284 2.9 3 F12.6A 316 320 10.7 11 3 285 284 3.0 3 F13.7B 365 368 14.0 13 6 282 284 2.9 3 F15.2A 275 275 8.0 8 3 317 314 4.1 4 J 290 290 9.0 9 3 340 344 4.9 5 MP143 322 317 11.1 11 3 280 284 2.9 3 USA232 368 368 14.2 15 1 312 314 3.9 4

1 The estimated number of RR1 repeats was calculated assuming 3 TN-repeats (i.e. the estimated length of the PCR product

subtracted with 155 bp and divided by the 15 bp of one repeat unit). 2 The RR1-repeat region is followed by a repeat-region of format GCT(ACTAAT)nACT on sequence level (or A(TN)nT on

amino acid level), whereby ‘n’ represents a number between 1 and 6. 3 The estimated number of RR2 repeats is determined by subtracting the estimated length of the amplified PCR product by

194 (i.e. the number of amplified base pairs not included in the repeat region) and dividing by 30 (i.e. the length of one

reiterated repeat unit).

Discussion In this study, M. hyopneumoniae isolates were differentiated by several typing techniques,

including some newly proposed and some techniques already described by other authors (1,

19, 35). Though multi-locus sequence typing (MLST) has been proposed as a key technique

to type and characterise strains of many bacterial species (31), it has not been worked out in

detail for M. hyopneumoniae. Moreover, MLST is still rather expensive to be used in routine

(22) and other molecular typing techniques may be favourable. However, these different

techniques were, until this study, never compared to each other for the typing of


M. hyopneumoniae. Based on results described here, we conclude that the techniques typed all

strains, but showed a different discriminatory power and reproducibility.

RAPD has been used as an easily performable and highly discriminatory test to type strains of

many Mycoplasma species (25). Also in our study, the discriminatory power was satisfactory,

despite the relatively low number of fragments using the described primer. On the other hand,

RAPD lacked reproducibility making a comparison with new isolates only possible by

reanalysing all isolates again in a single experiment. Such a low reproducibility has been

described before (e.g. 23, 33) and even for a species like M. gallisepticum, where RAPD has

often been successfully used for epidemiological studies, variation between gels and different

runs has been reported (12). Contrary to RAPD, AFLP yielded more complex banding

patterns and was much more reproducible. Although AFLP was reported fully reproducible

for mycoplasmas (19), conversion of AFLP patterns to gel images and subsequent analysis in

Bionumerics (Applied-Maths), yielded in our hands similarity values of 92% or higher for

replicates analysed on different days. Similar cut-off values have been reported for several

other bacterial species (e.g. 8, 32). This did not influence the discriminatory power of the

technique (>99%).

As demonstrated earlier (30), PFGE also proved to be a reproducible and highly

discriminatory typing technique. The use of two different restriction enzymes, SalI and ApaI,

was evaluated and yielded complementary results. The Simpson’s index of diversity for this

technique was calculated to be at least as high as 0.98, which is comparable to AFLP.

In this study, we additionally describe some molecular techniques that were never evaluated

for the typing M. hyopneumoniae. The VNTR of the P97 encoding gene were assessed.

Compared to the other methods, the estimation of the number of repeats in the P97 encoding

gene may be a fast and easily performable technique. Since it is PCR based, theoretically no

culture steps are necessary and the technique may give a first indication about possible

variation between two strains. However, a major drawback of the technique is its low

discriminatory power. Even when including the combined data of the two repeats, the

discriminatory power merely raised above 0.91. Moreover, the number of repeats can abruptly

change and more similar repeat units, preferably well-spread over the genome, should be

included before any postulations about the relation between strains can be made. With the

raising number of fully sequenced genomes and revelation of new regions with tandem

repeats, VNTR typing has been evaluated for and applied to several bacterial species (e.g. 24,

29, 36). This technique may especially be useful for mycoplasmas in general, which are


fastidious to cultivate and carry many repeats (26). However, in case of M. hyopneumoniae,

the number of genes containing tandem repeat regions appear to be limited (21). Moreover,

before setting up a VNTR typing scheme, the stability must be firmly validated since, in

contrast to our results for P97, many mycoplasma-specific proteins were reported to change

between different passages (27).

Despite the low discriminatory power, the use of P97 repeats in typing may be an indication

of the colonising capacities of the isolates. A direct link between the number of RR1 tandem

repeats and adhesion has been demonstrated and at least seven RR1 repeats seem to be

necessary to allow a strain to adhere to sodium dodecyl sulfate-solubilised porcine tracheal

cells in vitro (15). Strain F1.2A (BE) only contained two RR1 repeat regions. Still, the isolate

appears to be able to colonise the respiratory tract as it was isolated from a lung sample

collected in the slaughterhouse and was able to cause lesions in an experimental study (34).

This might indicate that besides P97 repeats, other colonisation factors may be present on

M. hyopneumoniae strains.

The P146 lipoprotein of M. hyopneumoniae shows a strong homology to the LppS lipoprotein

of M. conjunctivae, which was shown to be involved in in vitro adhesion (3). In addition, the

N-terminus region of P146 also shows a strong homology to the P97 adhesin (21) and

possesses a strong hydrophobic region (amino acid 7 –29), indicating a transmembrane region

and suggesting the protein to be expressed on the surface of M. hyopneumoniae cells. The

enormous intraspecific diversity shown for the P146 encoding gene was at least partly the

result of differences between several repeat regions present in the gene, most notably a poly-

serine chain of variable length and a [Q]n[(P/S)Q]m repeat region. Polyserine chains often

function as a spacer region in proteins involved in complex carbohydrate degradation (13),

while sequences rich in both proline and glutamine are not uncommon and can form a

conformation known as a polyproline II helix (18, 28). Such proline rich sequences are often

involved in binding processes and are highly immunogenic (18). Interestingly, Bencina et al.

(4) hypothesised a correlation between the length of a proline-rich region in the pvpA gene of

M. synoviae and virulence. Still, as long as the function of the P146 protein remains unknown,

correlations with virulence or adhesion are speculative and need further investigation.

In this study, the presence of size-variable regions increased the discriminatory power of the

P146 PCR-RFLP technique, which turned out to be very high (>0.98). Moreover, the typing

technique is reproducible and easy to perform. Similarly to estimation of the VNTR of P97,

the technique is PCR based and thus principally does not need pure cultures. The most


important drawback for epidemiological studies is the limited genomic region under

investigation. The molecular clock of the gene (i.e. the rate at which mutations are included)

is unknown and does not appear to be constant over the entire gene. The presence of the

highly variable regions seems in sharp contrast with the observed stability of the gene after

several in vitro passages and with the fact that several isolates of the same herd but of

different pigs had identical restriction patterns.

Apart from differences in reproducibility, complexity, and the differences in discriminatory

power, the different techniques yielded largely different clusters. Multiple isolates of herd

LH1 (LT), F15 (BE), F17 (BE) were indistinguishable with all described typing techniques,

including earlier described PFGE analyses (30), and likely represent a single clone. In

addition, isolates F21.9C (BE) and F23.7E (BE) were considered identical for all tests,

although the farms are from different geographical locations. Interestingly, the different

isolates of F16 were largely diverse for all techniques applied in this study, while they

appeared almost identical on the basis of PFGE analysis (30).

Although most techniques clustered the other isolates of the same farm in close proximity of

each other, not one technique uniquely clustered isolates according to their geographical

origin. Since an extensive variability was observed even for small geographical regions, an

association between country and isolate is unlikely, even by including more international

isolates. This complicates the comparison of different methods, since validation of typing data

is most effective when they are based on profound epidemiological knowledge and a known

phylogenetic relation between isolates. In case of M. hyopneumoniae, such a relation between

strains is hard to attain. Isolates are abundantly present in nature, highly diverse and often

cause infections that remain subclinical. In order to include at least some closely related

strains in our study, multiple isolates originating from the same herd were used. Our data

indeed indicate that mainly one clone is circulating at a specific point in time in a single herd.

It may be interesting to investigate the variability of isolates of the same farm over a longer

period of time to better understand and to model the transmission patterns of

M. hyopneumoniae clones. As demonstrated, various typing techniques with high

discriminatory power are available to perform such studies, although the use of more than one

technique may be essential to detect differences between closely related strains.

In conclusion, typing of M. hyopneumoniae can be easily performed with high discriminatory

power and reproducibility by restriction analysis of the highly variable gene encoding a P146

lipoprotein. However, the limited region under investigation may be insufficient to visualise


many genomic rearrangements. Therefore, AFLP or PFGE, although much more labour

intensive, may be preferred. RAPD lacks reproducibility, while determination of the number

of repeats in the gene encoding P97 may only be used as other epidemiological markers are

included as well. The different described techniques are useful to model the epidemiology of

M. hyopneumoniae, which may be helpful to develop precautionary measures in order to

control enzootic pneumonia in the future. Finally, a typing scheme containing P97 VNTR

analysis and P146 variability may be valuable for direct application on clinical samples and

might provide information on the adherence capacities or virulence of the strains. This should

however be further investigated.

Acknowledgements This study was supported by a grant of the Federal Service of Public Health, Food Chain


The authors are grateful to Dr. F. Friis, Dr. E. Thacker, Dr. A. van Essen, Dr. H. Windsor, and

Dr. K. Garlaite for providing us international M. hyopneumoniae isolates or lung samples. The

authors thank Sara Tistaert for skilful technical assistance.

References



2. Bashiruddin, J. B. 1998. Extraction of DNA from mycoplasmas. Methods Mol. Biol. 104:141-144.

3. Belloy, L., E. M. Vilei, M. Giacometti, and J. Frey. 2003. Characterization of LppS, an adhesin of

Mycoplasma conjunctivae. Microbiology. 149:185-193.

4. Bencina, D., M. Drobnic-Valic, S. Horvat, M. Narat, S. H. Kleven, and P. Dovc. 2001. Molecular

basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin. FEMS

Microbiol. Lett. 203:115-123.

5. Chen, J. W., L. Zhang, J. Song, F. Hwang, Q. Dong, J. Liui, and Y. Qian. 1992. Comparative

analysis of glycoprotein and glycolipid composition of virulent and avirulent strain membranes of

Mycoplasma hyopneumoniae. Curr. Microbiol. 24:189-192.


processing of the Mycoplasma hyopneumoniae cilium adhesin. Infect. Immun. 72:2791-2802.

7. Done, S. H. 1991. Environmental factors affecting the severity of pneumonia in pigs. Vet. Rec.

128:582-586.


8. Duim, B., T. M. Wassenaar, A. Rigter, and J. Wagenaar. 1999. High-resolution genotyping of

Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism

fingerprinting. Appl. Environ. Microbiol. 65:2369-2375.



10. Harasawa, R., K. Asada, and I. Kato. 1995. A novel repetitive sequence from Mycoplasma

hyopneumoniae. J. Vet. Med. Sci. 57:557-558.




43:145-156.

12. Hong, Y., M. Garcia, S. Levisohn, P. Savelkoul, V. Leiting, I. Lysnyansky, D. H. Ley, and S. H.

Kleven. 2005. Differentiation of Mycoplasma gallisepticum strains using amplified fragment length

polymorphism and other DNA-based typing methods. Avian Dis. 49:43-49.

13. Howard, M. B., N. A. Ekborg, L. E. Taylor, S. W. Hutcheson, and R. M. Weiner. 2004.

Identification and analysis of polyserine linker domains in prokaryotic proteins with emphasis on the

marine bacterium Microbulbifer degradans. Protein Sci. 13:1422-1425.

14. Hsu, T., S. Artiushin, and F. C. Minion. 1997. Cloning and functional analysis of the P97 swine

cilium adhesin gene of Mycoplasma hyopneumoniae. J. Bacteriol. 179:1317-1323.

15. Hsu, T., and F. C. Minion. 1998. Identification of the cilium binding epitope of the Mycoplasma

hyopneumoniae P97 adhesin. Infect. Immun. 66:4762-4766.

16. Hsu, T., and F. C. Minion. 1998. Molecular analysis of the P97 cilium adhesin operon of Mycoplasma

hyopneumoniae. Gene. 214:13-23.

17. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing

systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26:2465-2466.

18. Kay, B. K., M. P. Williamson, and M. Sudol. 2000. The importance of being proline: the interaction

of proline-rich motifs in signaling proteins with their cognate domains. FASEB J. 14:231-241.



20. Maes, D., H. Deluyker, M. Verdonck, F. Castryck, C. Miry, B. Vrijens, and A. de Kruif. 1999.

Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine influenza viruses and

Aujeszky's disease virus in slaughter pigs from fattening pig herds. Zentralbl. Veterinarmed. [B].

46:341-352.










24. Ramisse, V., P. Houssu, E. Hernandez, F. Denoeud, V. Hilaire, O. Lisanti, F. Ramisse, J. D.

Cavallo, and G. Vergnaud. 2004. Variable number of tandem repeats in Salmonella enterica subsp.

enterica for typing purposes. J. Clin. Microbiol. 42:5722-5730.

25. Rawadi, G. A. 1998. Characterization of mycoplasmas by RAPD fingerprinting. Methods Mol Biol.

104:179-187.



27. Rosengarten, R., and D. Yogev. 1996. Variant colony surface antigenic phenotypes within

mycoplasma strain populations: implications for species identification and strain standardization. J.


28. Rucker, A. L., and T. P. Creamer. 2002. Polyproline II helical structure in protein unfolded states:

lysine peptides revisited. Protein Sci. 11:980-985.

29. Scott, A. N., D. Menzies, T. N. Tannenbaum, L. Thibert, R. Kozak, L. Joseph, K. Schwartzman,

and M. A. Behr. 2005. Sensitivities and specificities of spoligotyping and mycobacterial interspersed

repetitive unit-variable-number tandem repeat typing methods for studying molecular epidemiology of

tuberculosis. J. Clin. Microbiol. 43:89-94.

30. Stakenborg, T., J. Vicca, P. Butaye, D. Maes, J. Peeters, A. D. Kruif, and F. Haesebrouck. 2005.

The diversity of Mycoplasma hyopneumoniae within and between herds using pulsed-field gel

electrophoresis. Vet. Microbiol. 109:29-36.



32. van Eldere, J., P. Janssen, A. Hoefnagels-Schuermans, S. van Lierde, and W. E. Peetermans.

1999. Amplified-fragment length polymorphism analysis versus macro-restriction fragment analysis for

molecular typing of Streptococcus pneumoniae isolates. J. Clin. Microbiol. 37:2053-2057.

33. Van Looveren, M., C. A. Ison, M. Ieven, P. Vandamme, I. M. Martin, K. Vermeulen, A. Renton,

and H. Goossens. 1999. Evaluation of the discriminatory power of typing methods for Neisseria

gonorrhoeae. J. Clin. Microbiol. 37:2183-2188.


34. Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. Haesebrouck. 2003.

Evaluation of virulence of Mycoplasma hyopneumoniae field isolates. Vet. Microbiol. 97:177-190.



36. Yazdankhah, S. P., B. A. Lindstedt, and D. A. Caugant. 2005. Use of variable-number tandem

repeats to examine genetic diversity of Neisseria meningitidis. J. Clin. Microbiol. 43:1699-1705.

171

CHAPTER IV

General Discussion

172 General Discussion

Introduction and definitions The general aims of this study involved the development of rapid, molecular tests for the

identification of Mollicutes species and for the typing of M. hyopneumoniae isolates. This

differentiation between species and/or isolates implies a good definition of an isolate, a clone

and a species. Therefore, at the start of this dissertation, we cautiously defined a strain or

isolate as a (sub)culture derived from a single pure colony, and the term clone as bacterial

cells that are indistinguishable in genotype at which the most likely explanation is a common

ancestor (I.2.1.1). Although these definitions seem straightforward, they are to a certain extent

dynamic. A single, bacterial cell that starts to replicate by binary fission should result in exact

copies of the same pure isolate. However, replication is never impeccable and, over time, a

variety of nearly identical isolates arise. Since differences between these isolates are very

small and frequently undetectable without applying whole genome sequence analysis, the

differences are considered negligible and the isolate is still regarded as pure. As replication

continues, strains further evolve and the number of differences between the ‘identical’ cells

augments. In practice, these cells will be able to spread into nature and they can,

subsequently, be isolated from different geographical areas or time frames. Still, as long as we

are unable to differentiate between these different isolates, they are described as one clone. At

a certain point of time (i.e. after a number of consecutive duplication events), a genotypic test

will enable us to visualize the differences between some of the replicates. At this point, the

isolates or clones are then to be considered as different. This logically implies that the

definition depends on the technique used to visualize differences. Noteworthy, in case the

technique is not fully reproducible, different laboratories may produce different results. When

extending this concept in terms of evolution (i.e. isolates able to replicate during millions of

years at a variety of different places), we might expect that a large number of different clones

will have arisen from one single ancestor at a rate that is largely dependent on the species

itself (22). Some clones may still be closely related, while others have become distantly

related. These latter clones may even be so different that they may form a new species,

implying that -similar to the definition of an isolate or clone- the definition of a species relies

on the ability and means to visualize differences. For Eukarya, the definition of a species is

rather straightforward and defined as a group of related organisms that are capable of

breeding with each other to produce fertile offspring (26). This definition is, however, hard to

maintain for prokaryotes that do not breed, but evolve by replication. Therefore, a bacterial

General Discussion 173

species may rather be defined as a clade (i.e. a monophyletic group that includes every

member of the group and its shared common ancestor) of organisms that show a high degree

of overall similarities (33). This species concept is a controversial issue and progresses with

the emergence of new methods. Consequently, an approved list of bacterial names is

periodically updated (11) and acknowledged species are regularly relocated as the term

species leaves room for debate (35). At the moment, a polyphasic approach is used to define

new species (43). This includes both phenetics (classify organisms based on overall similarity

regardless of their phylogeny or evolutionary relation using biochemical characteristics,

serological cross-reactions, DNA-DNA homology, etc.) and phylogenetics (reconstructing

evolutionary relationships usually on the basis of 16S rRNA gene sequences). Unfortunately,

some methods often yield divergent results, complicating the acknowledgement of new

species. Serology and DNA-DNA hybridisation studies may differ between different research

groups and species with comparable phenotypic characteristics or nearly identical 16S rRNA

gene sequences are common as well. Moreover, some species are primarily defined on the

basis of a striking phenotypic difference, usually related to the ability to cause disease (e.g.

Bacillus anthracis is in fact a clone of the free-living soil bacterium B. cereus) (14). At the

subspecies level, variation may be even more difficult to observe. Depending on experimental

conditions, largely variable values were obtained using DNA-DNA hybridisation studies for

M. mycoides subsp. mycoides LC and M. mycoides subsp. capri (75 to 94%), M. mycoides

subsp. mycoides LC and M. mycoides subsp. mycoides SC (88 to 93%), and M. mycoides

subsp. capri and M. mycoides subsp. mycoides SC (75 and 93%) (28). Sequence analysis of

16S rRNA genes revealed 99.9% similarity between M. mycoides subsp. mycoides LC and

M. mycoides subsp. capri (31). It goes without saying that the identification, defined as the

ability to discriminate between acknowledged species or subspecies (I.2.1.1), of such closely

related (sub)species is problematic and requires highly discriminatory identification

techniques. A polyphasic taxonomic approach, although essential for the designation of every

new species (43), is hard to sustain in practice for the identification of individual isolates.

Until this study, no unambiguously, satisfactory and generally applicable identification

techniques for Mycoplasma species had been described. Even molecular techniques based on

genotypic markers have often been considered inadequate (5). Numerous PCR tests with near-

perfect specificity have been described, but were not generally applicable.


Identification of Mollicutes species In this thesis, we examined the value of PCR amplification of the 16S rRNA gene using

universal primers in combination with restriction analysis (ARDRA) to differentiate

Mycoplasma species (III.1). Apart from being generally applicable, the technique has some

indisputable benefits. The technique is easy to perform, cost-effective and is highly

discriminatory. Even for closely related species with almost identical 16S rRNA gene

sequences, ARDRA is able to emphasize the few differences present without the need of

extensive 16S rDNA sequence analysis. The use of one restriction enzyme (AluI) could

distinguish no less than 73 out of 116 different Mycoplasma (sub)species. In combination

with BfaI or HpyF10VI, 31 additional species could be identified. Mycoplasmas contain 1 or

2 copies of the rrn operon and mutations in these copies may lead to unknown profiles,

although the chance of a single nucleotide mutation to fall in a restriction site is small. Even if

so, observed unknown profiles would seldom lead to misidentification, but rather complicate

analysis. Besides, obtained mixed, new patterns can be included in the identification scheme

and, if sufficiently stable, may even increase the discriminatory power of the technique. For

M. mycoides subsp. mycoides SC, characterized by a relatively high rate of 16S rRNA gene

micro-heterogeneity, differentiation on the basis of such a stable mutation was shown to be

very reliable (30, 31). A minor drawback of the technique is the imperfect size estimation.

Small differences between related profiles are sometimes hard to detect. Software tools may

be helpful for a rapid identification, although a visual confirmation using a positive control on

the same gel may be preferred. A parallel restriction with two restriction enzymes may help to

save time and ease interpretation as well, but will increase the overall costs of the technique.

For the technique to work on clinical samples, the use of Mycoplasma-specific instead of

universal primers might be worth considering. However, such previously described primers

were either too specific (4), not specific enough (8), or did amplify only a small region of the

16S rRNA gene, significantly decreasing the discriminatory power of the technique (42).

Possibly, the use of a mixture of primers may be desirable (46), though this should be further

examined. The fact that mixed profiles are not easily resolved might form an additional

shortcoming of the technique, especially in practice, since mycoplasmas tend to stick together

and filter cloning is a slow, drawn-out procedure.

To address some of these problems, a tDNA-PCR technique was developed (III.2). The

technique is based on the amplification of intergenic tDNA spacers by using outwardly


directed consensus tDNA primers. Like ARDRA, tDNA-PCR is rapid, easily performable,

and generally applicable. Unlike ARDRA, the technique comprises a high-resolution

electrophoresis step allowing exact size determination. Using a standardised protocol and

similar electrophoresis equipment (7), obtained fingerprints can be easily stored in a database.

This database can subsequently be used for the identification of unknown strains and allows

interlaboratory collaboration. Although different strains may have small differences in their

peak profiles, the method has been proven reliable for a rapid identification. Only two closely

related species (M. bovis and M. agalactiae) could not be distinguished and for inexplicable

reasons, M. gallisepticum did not yield a fingerprint. In our study, dealing with a historic

collection, it was shown that in several occasions mixed samples of two species could be

resolved. Moreover, our results demonstrated that tDNA-PCR may be useful for the direct

detection and identification of the extremely fastidious M. genitalium in clinical samples.

However, when working with clinical samples, the DNA preparation step is of vital

importance and remains, as with all PCR techniques, an important bottleneck for detection

(32). PCR assays performed directly on clinical samples are still not considered reliable for

detection since inhibitors often lead to false negative results or low sensitivity, while false

positive results occur frequently as a result of laboratory and aerosol contaminations (44). So,

even if the sample preparation is optimised, PCR assays must be firmly validated by parallel

testing using other methods. Logically, most bacterial species, which are far less fastidious to

cultivate, are mainly diagnosed by pure culture. It is not only cheap and reliable; isolates can

be used for typing or directly be screened for antibiotic resistance to optimise treatment. For

other, more fastidious bacterial organisms such as mycoplasmas, there is a pressing need for

the introduction of standardized molecular techniques in routine practice. In our study,

generally applicable, easily performable tests to correctly identify isolates of several genera

belonging to the class of Mollicutes were developed. These techniques proved already

valuable for the identification of several genera, mycoplasmas in particular, but the usefulness

of the techniques to identify all Mollicutes species, especially the non-cultivable

phytoplasmas, should be further examined.


Isolation, identification and typing of M. hyopneumoniae strains After development of generally applicable identification techniques, we focused on the

isolation, identification and typing of M. hyopneumoniae in particular. M. hyopneumoniae is

the major cause of enzootic pneumonia in pigs, but not much is known about its virulence

mechanism, neither about the population structure of this organism. Some molecular typing

techniques have been described for M. hyopneumoniae, but have never been used extensively,

mainly because of the limited number of isolates available for research. Therefore, to set-up a

collection, M. hyopneumoniae strains were isolated from lungs of slaughter pigs according to

a method described by Friis (13). This isolation procedure has been developed more than 30

years ago and has never been changed since. Perhaps others may have tried, but never

succeeded to improve the isolation of this fastidious organism. Isolation takes at least several

weeks and small, nearly visible colonies only grow on solid agar after several passages in

broth culture. This need of adaptation for colonies to grow on solid agar, underlines the

difficulties for reproducing an ideal micro-environment for growth.

Friis’ medium is a rich broth, and other porcine mycoplasmas, namely M. flocculare and

M. hyorhinis, are frequently co-isolated. Since M. hyorhinis grows much faster, its presence

may seriously compromise the isolation of M. hyopneumoniae. The closely related

M. flocculare, on the other hand, grows as slow as M. hyopneumoniae and may be difficult to

distinguish (38). To simplify the identification steps during isolation, a multiplex PCR for

simultaneously differentiating these three species was developed (III.3). This method does not

only directly identify M. hyopneumoniae, it also verifies the absence of the other two porcine

mycoplasmas in the (pure) culture. A fourth porcine mycoplasma, namely M. hyosynoviae,

was not included, since it ferments arginine and does not grow in Friis’ broth.

In this manner, M. hyopneumoniae strains from over 20 herds were isolated and identified in

our laboratory. Moreover, multiple strains from a single farm were isolated. This relatively

large collection of M. hyopneumoniae isolates, supplemented with some international and

reference strains, enabled us to perform and develop some typing techniques as a basis for

future molecular epidemiological studies. The need of a diverse collection is essential to

perform such experiments, since one organism unlikely represents the multitude of strains

forming a ‘species’ (6).

Epidemiological studies will not only provide the means to determine the heterogeneity of the

species, it may also allow to delineate certain subgroups (clonal lineages) within a species, for


instance according to their geographical origin or date of isolation. In an ideal typing method,

all epidemiologically related isolates yield identical fingerprints, different from those derived

from other isolates of the same species. In other words, included markers should be

sufficiently stable (over a limited period of time) for related isolates to yield identical

fingerprints, while they must be variable enough (over a longer period of time) to reflect the

diversity within a species (23). However, not all genetic events occur at the same frequency

and different typing systems may be chosen to answer different epidemiological questions.

Indeed, frequently, one technique can cluster strains according to larger groups (a certain host,

geographical region), while other techniques, possibly with a greater discriminatory power,

are not able to do so.

A typing system preferentially aims to visualize the true phylogenetic relationships between

strains, though it may be difficult to avoid that also some less related strains may yield similar

fingerprints, depending on the discriminatory power of the technique used. A strain may differ

from its ancestor not only as a result of nucleotide mutations, genomic rearrangements, gene

loss, or gene duplication; also horizontal gene transfer may play an important role (1, 6). In

contrary to Eukarya, where speciation is the result of events that prohibit the exchange of

genomic fragments, Bacteria are capable of interspecific gene transfer. Although detailed

studies about this issue are to our knowledge still lacking for Mollicutes, the difference in

genetic code may indicate that Mollicutes participate less actively in gene exchange with other

bacterial classes. The failure to find drug resistance plasmids in mycoplasmas partially

supports this hypothesis (26). Between mycoplasmas themselves, horizontal gene transfer

may be more important, particularly for mobile elements such as insertion sequences (12, 40).

Further studies on horizontal gene transfer may show that single strains may have inherited

DNA from different parental strains and the current phylogenetic trees may need to be

replaced by complex 3-dimensional networks in order to fully visualize the true phylogeny

(21). Full genomic sequences of different strains of one species will permit to reconstruct and

better understand the true evolution of a species.

Ideally, useful typing techniques must be reproducible to allow interlaboratory comparisons,

have a high discriminatory power, be performable on preferentially all strains (i.e.

typeability), be easy to perform and interpret, and if possible be cheap as well. It is extremely

hard to find one technique that qualifies for all these aspects (III.5). PFGE was, until recently,

promoted as a highly reproducible and discriminatory technique, representing a gold standard

for typing (29, 39). However, PFGE is being replaced by MLST for many important


pathogens. The latter technique is based on the determination of partial nucleotide sequences

of several conserved (housekeeping) genes, which are widespread over the genome (41).

MLST is found beneficial because the generated data are highly discriminatory, can be easily

compared with those of other laboratories, and can be used to calculate the phylogenetic

relationship between strains for the construction of an evolutionary tree. An MLST scheme

has not been worked out for M. hyopneumoniae yet, and PFGE or AFLP may still be effective

and less costly alternatives. The disadvantage of these two techniques is their requirement for

pure cultures, which are cumbersome to obtain. Therefore, some PCR based typing methods

were designed and/or examined as well. RAPD was unsuited due to its low reproducibility,

even on purified genomic and carefully quantified DNA. Determination of the VNTR of P97

proved unsatisfactory because of a low discriminatory power. PCR-RFLP of a highly variable

P146 gene, on the other hand, has a high discriminatory power and reproducibility, but, since

the technique only explores a small fraction of the genome, it is unlikely representative for the

diversity of the entire genome. Nevertheless, the use of these latter PCR-based typing

methods is very promising. Especially for Mycoplasma species, a direct application on

clinical material or on low numbers of bacteria would be advantageous since it omits at least

in part the difficult isolation procedures. Besides, there are indications that such variable

genes may be directly linked to adherence or other phenotypic characteristics. This however

needs further investigation. A direct link between the number of RR1 tandem repeats of P97

and adhesion has already been demonstrated (18). Remarkably, one isolate (F1.2A) had only

two RR1 repeats, while at least seven RR1 repeats are expected to be necessary for adhesion

(18). Possibly, the varying number of observed RR2- or TN-repeats with unknown function

may have an influence on attachment, while also other proteins, such as P146, may be

involved in adherence as well. P146 shows both homology to the adhesin P97 and the LppS

lipoprotein of M. conjunctivae, which has been correlated to the attachment to joint synovial

cells in vitro (2). The P146 protein is especially fascinating because it contains some highly

variable regions, most notably a polyserine chain and a proline rich region. Polyserine chains

have most often been found in proteins involved in complex carbohydrate degradation where

they function as a spacer region, presumably to optimise substrate accessibility (17).

Sequences rich in proline and glutamine, on the other hand, are often involved in binding

processes and are highly immunogenic (19). The pvpA gene of M. synoviae, which is rich in

proline residues as well, was even hypothetically correlated with virulence (3).


However, the function of the P146 protein is still unknown and urges for more research.

Unfortunately, the absence of cloning and expression vectors greatly complicates such

studies. Mycoplasmas have a different codon usage making standard gene-technological

methods impossible to use. Moreover, attempts to transform M. hyopneumoniae were so far

unsuccessful and research projects to cope with these problems should be initiated, especially,

since enzootic pneumonia remains an economically important disease.

In no less than 30 to 80% of the piggeries worldwide, clinical signs related to

M. hyopneumoniae infections are observed (20). Studies indicate that direct contact between

pigs are the main source of infection, although airborne transmission up to over 3 kilometres

(9, 15) may occur as well and lead to the (re)infection of specific pathogen free farms (16,

37). Our results showed that highly different clones were observed in different farms. Even

clones carrying antibiotic resistance markers seemed largely different and unlikely to spread

between farms (36, 45), but seem to originate independently by means of acquiring point

mutations. In contrast with this diversity, our results indicate that at one particular moment in

time, especially one specific clone (or a limited number of related clones) is circulating in a

single farm. This raises questions about the way M. hyopneumoniae strains spread between

farms and about the rate at which new clones emerge and disappear in nature. Further

epidemiological studies, preferably with serial samples of different age groups and of a

geographically close entity, should be carried out to answer these unresolved questions.

The divergence between M. hyopneumoniae isolates in vitro was at least supported by our

PFGE data (III.4), which showed a large genomic inversion in reference strain 232 when

compared to its genome sequence (27). Furthermore, the variability observed in the P97 and

P146 genes indicate a great genomic plasticity for M. hyopneumoniae strains. Similarly,

highly variable genes were observed in other Mycoplasma species as well. The frequency of

phase-transition of vsp and vsa genes of M. bovis and M. pulmonis, respectively, was

estimated to be about 10-3 per cell per generation (25, 34). In fact, most mycoplasmas seem to

have an elevated spontaneous mutation rate as was first observed for the ribosomal RNA gene

sequences, which have drifted further compared to other bacterial lines (47). All these events

place mycoplasma genomes among the most variable known, although it must be said that

Mycoplasma species with relatively stable genomes have been reported as well (10, 24).

In conclusion, since traditional tests are limited in the differentiation of strains, molecular

tests are indispensable. Over time, their importance will likely further expand. Keeping in

mind the enormous diversity of strains within a species, it may become important not only to


identify the species, but also rather to determine which clone is causing a disease. To this end,

the gap between genotype and phenotype should be closed, which is without doubt one of the

great tasks of 21st century. With such future research and expertise, the virulence mechanisms

of M. hyopneumoniae may eventually be fully elucidated. Nevertheless, scientists will

definitely run into other challenges in the field of mycoplasmology. The number of

Mycoplasma species and hosts is that diverse, that one-day or the other (new) mycoplasmas

will appear where we might not expect to find them.

References

1. Arber, W. 2000. Genetic variation: molecular mechanisms and impact on microbial evolution. FEMS

Microbiol. Rev. 24:1-7.

2. Belloy, L., E. M. Vilei, M. Giacometti, and J. Frey. 2003. Characterization of LppS, an adhesin of

Mycoplasma conjunctivae. Microbiology 149:185-193.

3. Bencina, D., M. Drobnic-Valic, S. Horvat, M. Narat, S. H. Kleven, and P. Dovc. 2001. Molecular

basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin. FEMS

Microbiol. Lett. 203:115-123.

4. Blanchard, A., M. Gautier, and V. Mayau. 1991. Detection and identification of mycoplasmas by

amplification of rDNA. FEMS Microbiol. Lett. 65:37-42.



6. Coenye, T., D. Gevers, Y. Van de Peer, P. Vandamme, and J. Swings. 2005. Towards a prokaryotic

genomic taxonomy. FEMS Microbiol. Rev. 29:147-167.

7. De Baere, T., A. Van Keerberghen, P. Van Hauwe, H. De Beenhouwer, A. Boel, G. Verschraegen,

G. Claeys, and M. Vaneechoutte. 2005. An interlaboratory comparison of ITS2-PCR for the

identification of yeasts, using the ABI Prism 310 and CEQ8000 capillary electrophoresis systems. BMC

Microbiol. 5:14.




9. Done, S. H. 1991. Environmental factors affecting the severity of pneumonia in pigs. Vet. Rec.

128:582-586.

10. Dumke, R., I. Catrein, E. Pirkil, R. Herrmann, and E. Jacobs. 2003. Subtyping of Mycoplasma

pneumoniae isolates based on extended genome sequencing and on expression profiles. Int. J. Med.

Microbiol. 292:513-525.


11. Euzeby, J. P., L. Hoyles, P. Kampfer, A. Oren, G. S. Saddler, H. G. Truper, and B. J. Tindall.

2004. 'List of changes in taxonomic opinion': making use of the new lists. Int. J. Syst. Evol. Microbiol.

54:1429-1430.

12. Ferrell, R. V., M. B. Heidari, K. S. Wise, and M. A. McIntosh. 1989. A mycoplasma genetic element

resembling prokaryotic insertion sequences. Mol. Microbiol. 3:957-967.


and Mycoplasma flocculare: a survey. Nord. Vet. Med 27:337-339.

14. Gevers, D., F. M. Cohan, J. G. Lawrence, B. G. Spratt, T. Coenye, E. J. Feil, E. Stackebrandt, Y.

V. de Peer, P. Vandamme, F. L. Thompson, and J. Swings. 2005. Opinion: Re-evaluating

prokaryotic species. Nat. Rev. Microbiol. 3:733-739.

15. Goodwin, R. F. 1972. Experiments on the transmissibility of enzootic pneumonia of pigs. Res. Vet.

Sci. 13:257-261.




43:145-156.

17. Howard, M. B., N. A. Ekborg, L. E. Taylor, S. W. Hutcheson, and R. M. Weiner. 2004.

Identification and analysis of polyserine linker domains in prokaryotic proteins with emphasis on the

marine bacterium Microbulbifer degradans. Protein Sci. 13:1422-1425.

18. Hsu, T., and F. C. Minion. 1998. Identification of the cilium binding epitope of the Mycoplasma

hyopneumoniae P97 adhesin. Infect. Immun. 66:4762-4766.

19. Kay, B. K., M. P. Williamson, and M. Sudol. 2000. The importance of being proline: the interaction

of proline-rich motifs in signaling proteins with their cognate domains. FASEB J. 14:231-241.

20. Kobisch, M., and N. F. Friis. 1996. Swine mycoplasmoses. Rev. Sci. Tech 15:1569-1605.

21. Kunin, V., L. Goldovsky, N. Darzentas, and C. A. Ouzounis. 2005. The net of life: Reconstructing

the microbial phylogenetic network. Genome Res. 15:954-959.

22. Lawrence, J. G. 2001. Catalyzing bacterial speciation: correlating lateral transfer with genetic

headroom. Syst. Biol. 50:479-496.

23. Lipuma, J. J. 1998. Molecular tools for epidemiologic study of infectious diseases. Pediatr. Infect. Dis.

J. 17:667-675.

24. Lorenzon, S., I. Arzul, A. Peyraud, P. Hendrikx, and F. Thiaucourt. 2003. Molecular epidemiology

of contagious bovine pleuropneumonia by multilocus sequence analysis of Mycoplasma mycoides

subspecies mycoides biotype SC strains. Vet. Microbiol. 93:319-333.


25. Lysnyansky, I., R. Rosengarten, and D. Yogev. 1996. Phenotypic switching of variable surface

lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. J. Bacteriol.

178:5395-5401.

26. Meyer, A. 2005. On the importance of being Ernst Mayr. PLoS Biol. 3:e152.




28. Monnerat, M. P., F. Thiaucourt, J. Nicolet, and J. Frey. 1999. Comparative analysis of the lppA

locus in Mycoplasma capricolum subsp. capricolum and Mycoplasma capricolum subsp.

capripneumoniae. Vet. Microbiol. 69:157-172.

29. Murchan, S., M. E. Kaufmann, A. Deplano, R. de Ryck, M. Struelens, C. E. Zinn, V. Fussing, S.

Salmenlinna, J. Vuopio-Varkila, N. El Solh, C. Cuny, W. Witte, P. T. Tassios, N. Legakis, W. van

Leeuwen, A. van Belkum, A. Vindel, I. Laconcha, J. Garaizar, S. Haeggman, B. Olsson-Liljequist,

U. Ransjo, G. Coombes, and B. Cookson. 2003. Harmonization of pulsed-field gel electrophoresis

protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single

approach developed by consensus in 10 European laboratories and its application for tracing the spread

of related strains. J. Clin. Microbiol. 41:1574-1585.




3821.



genes from the two rRNA operons. J. Bacteriol. 178:4131-4142.



33. Rossello-Mora, R., and R. Amann. 2001. The species concept for prokaryotes. FEMS Microbiol. Rev.

25:39-67.

34. Simmons, W. L., C. Zuhua, J. I. Glass, J. W. Simecka, G. H. Cassell, and H. L. Watson. 1996.

Sequence analysis of the chromosomal region around and within the V-1-encoding gene of Mycoplasma

pulmonis: evidence for DNA inversion as a mechanism for V-1 variation. Infect. Immun. 64:472-479.

35. Stackebrandt, E., W. Frederiksen, G. M. Garrity, P. A. D. Grimont, P. Kämpfer, M. C. J.

Maiden, X. Nesme, R. Rossello-Mora, J. Swings, H. G. Trüper, L. Vauterin, A. C. Ward, and W.

B. Whitman. 2002. Report of the ad hoc committee for the reevaluation of the species definition in

bacteriology. Int. J. Syst. Evol. Microbiol. 52:1043-1047.


36. Stakenborg, T., J. Vicca, P. Butaye, D. Maes, F. C. Minion, J. Peeters, A. de Kruif, and F.

Haesebrouck. 2005. Characterization of in vivo acquired resistance of Mycoplasma hyopneumoniae to

macrolides and lincosamides. Microb. Drug Resist. 11:291-295.

37. Stärk, K. D. 2000. Epidemiological investigation of the influence of environmental risk factors on

respiratory diseases in swine--a literature review. Vet. J. 159:37-56.






7:382-389.

40. Thomas, A., A. Linden, J. Mainil, D. F. Bischof, J. Frey, and E. M. Vilei. 2005. Mycoplasma bovis

shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides

SC: Evolutionary and developmental aspects. FEMS Microbiol. Lett. 245:249-255.



42. van Kuppeveld, F. J., J. T. van der Logt, A. F. Angulo, M. J. van Zoest, W. G. Quint, H. G.

Niesters, J. M. Galama, and W. J. Melchers. 1992. Genus- and species-specific identification of

mycoplasmas by 16S rRNA amplification. Appl. Environ. Microbiol. 58:2606-2615.

43. Vandamme, P., B. Pot, M. Gillis, P. de Vos, K. Kersters, and J. Swings. 1996. Polyphasic

taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60:407-438.

44. Vaneechoutte, M., and J. Van Eldere. 1997. The possibilities and limitations of nucleic acid

amplification technology in diagnostic microbiology. J. Med. Microbiol. 46:188-194.

45. Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. Haesebrouck. 2004. In

vitro susceptibilities of Mycoplasma hyopneumoniae field isolates. Antimicrob. Agents Chemother.

48:4470-4472.

46. Wirth, M., E. Berthold, M. Grashoff, H. Pfutzner, U. Schubert, and H. Hauser. 1994. Detection of

mycoplasma contaminations by the polymerase chain reaction. Cytotechnology 16:67-77.

47. Woese, C. R., E. Stackebrandt, and W. Ludwig. 1984. What are mycoplasmas: the relationship of

tempo and mode in bacterial evolution. J. Mol. Evol. 21:305-316.

185

SUMMARY

Mollicutes, characterised by the absence of a cell wall, most likely arose from the

Streptococcus phylogenetic branch about 600 million years ago. During their degenerative

evolution, they lost many genes involved in anabolic metabolism. To compensate for this loss,

they reside as obligatory parasites in an extensive number of plant and animal hosts with a

manifold of nutrients at hand. Such an environment is hard to mimic and in vitro cultivation

of these fastidious bacteria is difficult to attain. Recovery of mycoplasmas by culture

generally takes several weeks, and when successful, identification is another problem to cope

with. Classical tests, mainly performed on pure colonies, tend to fall short because serological

tests often yield misleading results, and biochemical tests are not discriminative enough for

interspecific differentiation. With the enormous number of described Mollicutes species,

laboratories are generally specialised in the identification of only a number of important

pathogens, while other mycoplasmas often remain neglected. Consequently, new methods for

rapid laboratory diagnosis based on nucleic acid amplification techniques are increasingly

important.

Therefore, we developed two generally applicable molecular techniques for identification

purposes. In the first method, the applicability of amplified rDNA restriction analysis

(ARDRA) for the identification of acknowledged Mycoplasma species and subspecies was

examined. Based upon available 16S rDNA sequences, theoretical ARDRA profiles were

calculated and their discriminatory power was determined. Restriction endonuclease AluI

(AG^CT) was found to be highly discriminatory and was used alone or in combination with

BfaI (C^TAG) or HpyF10VI (GCNNNNN^NNGC) to identify almost all Mycoplasma

species. The in silico determined patterns were verified on 60 strains of 27 different species

and subspecies. All in vitro obtained restriction profiles were in accordance with the

calculated fragments based on only one 16S rDNA sequence, except for two isolates of M.

columbinum and two isolates of the M. mycoides cluster, for which correct ARDRA profiles

were only obtained if the sequences of both rrn operons were taken into account.

In the second method, the applicability of tDNA-PCR was examined for the identification of

different Mollicutes species. Reproducible peak profiles were generated for a total of 91 out of

103 DNA extracts belonging to 30 different species of the genera Acholeplasma, Mycoplasma

186 Summary

and Ureaplasma. Of the 12 failures, nine were likely the result of inapt DNA, while the

failure of the other three samples, all M. gallisepticum, was less clear. Six other samples

yielded a mixed pattern, while one had a unique pattern. The 16S rRNA gene sequence of this

latter isolate did not match with any of the published sequences, pointing to the existence of a

not yet described species.

In conclusion, we found both ARDRA and tDNA-PCR to be rapid and discriminatory

methods to correctly identify a large collection of different species of the class of Mollicutes.

In a second part of this study, we investigated the diversity of M. hyopneumoniae isolates by

different molecular typing techniques. An isolation procedure was set up. To optimise the

isolation, a multiplex PCR was developed as a helpful tool for a rapid differentiation of

M. hyopneumoniae, M. hyorhinis and M. flocculare, since these three mycoplasma species all

require similar growth conditions and can be recovered simultaneously. After choosing a set

of specific primers and optimising reaction conditions, specific PCR products were observed

for each of the three species. The amplicons differed in size (1129 bp for M. hyorhinis, 1000

bp for M. hyopneumoniae, and 754 bp for M. flocculare) and were clearly distinguishable on a

1% agarose gel. Together with the use of this technique, a collection of M. hyopneumoniae

isolates originating from over 20 herds was obtained. This collection, supplemented with

several international and reference strains, was used to compare different typing techniques

for their value and accuracy. Amplified fragment length polymorphism (AFLP) and random

amplified polymorphic DNA (RAPD) analysis were described before, while pulsed-field gel

electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) of the gene

encoding lipoprotein P146, and the variable number of reiterated repeats (VNTR) of the P97

encoding gene were for the first time used as possible methods to type M. hyopneumoniae

isolates. All techniques, except PFGE, showed a typeability of 100% and demonstrated a high

intraspecific diversity. However, the discriminatory power of the different techniques varied

considerably. AFLP (>0.99), PFGE (>0.98), and PCR-RFLP of the P146 encoding gene

(>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97

(<0.92). RAPD was also found a less interesting typing technique because of its low

reproducibility between different runs.

All molecular techniques showed overall more resemblance between strains isolated from

different pigs originating from a same herd. These results indicate a closer relationship of

M. hyopneumoniae isolates originating from within a herd compared to isolates from different

Summary 187

herds. On the other hand, for these latter isolates originating from different farms, none of the

techniques was able to show a clear relationship between the geographical origin and the

obtained fingerprints.

We conclude that AFLP and PFGE are highly reliable and discriminatory typing techniques to

outline the genomic diversity of M. hyopneumoniae isolates. Our data also show that RFLP of

a highly variable gene encoding P146 may be a useful alternative to demonstrate intraspecific

variability, although the generation of sequence variability of the gene remains unclear and

must be further examined.

189

SAMENVATTING

De Mollicutes, die gekenmerkt worden door de afwezigheid van een celwand, vormen een

speciale klasse van bacteriën. Ze zijn naar alle waarschijnlijk 600 miljoen jaar geleden

ontstaan uit een fylogenetische tak van de streptokokken. Gedurende hun verdere evolutie

hebben ze veel van hun genen, betrokken bij anabolisme, verloren. Het is daarom niet te

verwonderen dat mollicuten als parasieten te vinden zijn bij talloze planten en dieren alwaar

de voor hen noodzakelijke voedingsstoffen gemakkelijk beschikbaar zijn. Een dergelijk rijk

milieu is moeilijk na te bootsen in vitro en hun isolatie verloopt dikwijls moeizaam.

Daarenboven is, naast de moeilijke cultivatie, ook de identificatie van Mollicutes species

veelal problematisch. Klassieke identificatie testen, meestal gebaseerd op zuivere kolonies,

zijn zelden bruikbaar omdat enerzijds serologische testen dikwijls kruisreageren, terwijl

anderzijds biochemische testen meestal onvoldoende discriminerend zijn. Dit wordt nog extra

bemoeilijkt door het enorme aantal Mollicutes species. Het gevolg is dat laboratoria dikwijls

gespecialiseerd zijn in de isolatie en identificatie van slechts enkele belangrijke pathogenen,

terwijl tal van andere species genegeerd worden. Dit verklaart de grote nood aan snelle,

nieuwe technieken om de identificatie van Mollicutes species te vergemakkelijken.

Twee nieuwe, algemeen toepasbare identificatietechnieken werden ontwikkeld. In de eerste

plaats werd de bruikbaarheid van amplified rDNA restriction analysis (ARDRA) nagegaan

voor de identificatie van de erkende Mycoplasma species en subspecies. Gebaseerd op

beschikbare 16S rDNA sequenties werden de ARDRA profielen theoretische berekend en het

discriminerend vermogen van de techniek bepaald. Restrictie endonuclease AluI (AG^CT)

bleek sterk discriminerend en werd op zich, of in combinatie met BfaI (C^TAG) of HpyF10VI

(GCNNNNN^NNGC), gebruikt om nagenoeg alle Mycoplasma species en subspecies te

identificeren. De in silico berekende restrictie patronen werden bovendien gecontroleerd voor

60 stammen behorende tot 27 verschillende species en subspecies. Met uitzondering van vier

isolaten, kwamen alle in vitro patronen overeen met de theoretisch bepaalde. De vier

uitzonderingen, zijnde twee M. columbinum isolaten en twee isolaten behorende tot de M.

mycoides cluster, vertoonden meer restrictiefragmenten dan verwacht. Dit bleek het gevolg

van sequentieverschillen in de twee kopijen van de in het genoom aanwezige rRNA genen.

190 Samenvatting

Enkel bij het in rekening brengen van beide sequenties kwamen de theoretisch bepaalde en in

vitro bekomen restrictieprofielen overeen.

Vervolgens werd de bruikbaarheid van een tDNA-PCR nagegaan voor de identificatie van

verscheidene Mollicutes species. Reproduceerbare piekprofielen werden bekomen voor 91

van de 103 onderzochte DNA extracten afkomstig van 30 verschillende species van de genera

Acholeplasma, Mycoplasma en Ureaplasma. Voor 12 DNA extracten werden geen piek-

profielen bekomen. In 9 gevallen was dit te wijten aan een slechte kwaliteit van het gebruikte

DNA terwijl voor 3 andere stammen, allen M. gallisepticum, er geen duidelijke verklaring

voor het negatief resultaat werd gevonden. Zes andere stalen gaven een gemengd piekpatroon,

terwijl voor één stam een uniek profiel bekomen werd. Sequentie analyse van het 16S rRNA

gen van dit isolaat kwam niet overeen met reeds bekende sequenties. Dit duidt er op dat het

hier een nieuw, niet eerder beschreven, species betreft.

Er kon besloten worden dat zowel ARDRA als tDNA-PCR snelle en sterk discriminerende

technieken bleken te zijn, bruikbaar voor de correcte identificatie van een groot aantal

verschillende Mollicutes species.

Het tweede deel van dit doctoraat had als doel de diversiteit van M. hyopneumoniae isolaten

te onderzoeken. Daartoe werden verschillende moleculaire technieken ingezet. Vooraf

dienden verschillende M. hyopneumoniae isolaten bekomen te worden uit het

ademhalingssstelsel van varkens. Om de identificatie van deze kiem tijdens de isolatie te

vereenvoudigen werd een multiplex PCR ontwikkeld. Deze multiplex PCR laat een snelle en

simultane identificatie van M. hyopneumoniae, M. hyorhinis en M. flocculare in

cultuurmedium toe. Na het kiezen van een set van specifieke primers en optimalisatie van de

PCR reactie condities, werd na amplificatie voor elk species een specifiek fragment bekomen.

De PCR producten verschilden in grootte (1129 bp voor M. hyorhinis, 1000 bp voor M.

hyopneumoniae, en 754 bp voor M. flocculare) en kunnen duidelijk onderscheiden worden op

een 1% agarose gel. Met behulp van deze techniek werden M. hyopneumoniae isolaten

geïdentificeerd tijdens de isolatie van stammen afkomstig van meer dan 20 verschillende

bedrijven.

Deze collectie, aangevuld met verschillende internationale isolaten en referentiestammen,

werd gebruikt om de waarde van verschillende beschreven en enkele nieuw ontwikkelde

moleculaire typeringstechnieken met elkaar te vergelijken en in te schatten. Amplified

fragment length polymorphism (AFLP) en randomly amplified polymorphic DNA (RAPD)

Samenvatting 191

analyses werden reeds eerder beschreven, terwijl pulsed-field gelelectrophoresis (PFGE),

restriction fragment length polymorphism (RFLP) van het gen coderend voor een P146

lipoproteïne en de bepaling van het variabel aantal tandem repeats (VNTR) aanwezig in het

gen coderend voor het P97 adhesine voor de eerste maal gebruikt werden als

typeringstechniek voor M. hyopneumoniae. Alle technieken, met uitzondering van PFGE,

vertoonden een typeerbaarheid van 100% en toonden allen een grote heterogeniteit van de

isolaten aan. Het discriminerend vermogen van de verschillende testen varieerde daarentegen

aanzienlijk. AFLP (>0.99), PFGE (>0.98), en PCR-RFLP van het P146 gen (>0.98) waren

meer discriminerend dan RAPD (0.95) en bepaling van het VNTR van P97 (<0.92). RAPD

was bovendien een minder interessante typeringstechniek wegens zijn lage

reproduceerbaarheid tussen verschillende experimenten.

Alle technieken toonden meer gelijkenissen aan tussen isolaten afkomstig van één bedrijf in

vergelijking met isolaten afkomstig van verschillende bedrijven, wat aanduidt dat isolaten

afkomstig van eenzelfde bedrijf nauwer verwant zijn. Met geen enkele techniek werd een

duidelijk verband gevonden tussen het land van herkomst en het bekomen typeringsprofiel.

Uit deze resultaten bleek dat AFLP en PFGE betrouwbare en sterk discriminerende

technieken zijn om de genomische variabiliteit van M. hyopneumoniae isolaten aan te tonen.

De bekomen data duiden ook op de bruikbaarheid van PCR-RFLP van het sterk variërend

P146 gen als alternatief, hoewel de mutatiesnelheid van dit laatste gen verder onderzocht

moet worden.

193

DANKWOORD

De dag ontwaakt, de zonne zonk, het duister klom. Na uren (dagen, maanden) als het ware

vastgekluisterd achter mijn PC, zijn dit de laatste en ongetwijfeld belangrijkste woorden van

mijn doctoraatsthesis.

En als er zoveel mensen zijn die je wilt en moet bedanken, waar dan beginnen? Misschien bij

Dr. Jan Mast en Jonas, die me als eersten wegwijs maakten in the obscure world of research

where small things still matter (en deze keer spreek ik niet enkel van micro-organismen en

DNA). Dan zijn er vooreerst ook een hele reeks mensen die me tijdens het schrijven van dit

doctoraat op wetenschappelijk gebied ondersteund hebben. De meesten onder hen zijn reeds

vermeld als co-auteur of staan reeds opgenomen in de acknowledgements. Desalniettemin zou

ik sommigen van hen graag nog eens extra in het licht plaatsen: Jo voor de altijd -ondanks de

afstand- vlotte samenwerking; Lies voor de hulp bij de opstart (of beter ‘opgroei’) van het

mycoplasma-project; Dr. Patrick Butaye voor de immer blijvende steun en voor de prioriteit

die hij telkens aan mijn werk wist te geven ondanks zijn immer rinkelende telefoon; Prof. Dr.

Dominiek Maes voor de nauwe, steeds correcte opvolging en zijn gedrevenheid; Prof. Dr. F.

Haesebrouck voor de final touch die de teksten toch altijd net dat ietsje beter maakten en zijn

onmisbare raadgevingen; Dr. Johan Peeters, Dr. Hein Imberechts en Prof. Dr. A. de Kruif, die

ondanks hun drukke agenda steeds aanspreekbaar en betrokken bleven; Prof. Dr. Mario

Vaneechoutte voor de toffe mails en vlotte samenwerking die een blijvende stimulans

vormden bij het schrijven van grote stukken van dit doctoraat; Saar, Véronique, evenals de

andere laboranten voor hun hulp bij de praktische uitvoering van experimenten; de leden van

de lees- en examencommissie voor hun constructieve opmerkingen en de tijd die ze

vrijmaakten om mijn thesis te lezen en te beoordelen. Kortom, graag zou ik alle collega’s die

meehielpen bij de voltooiing van dit doctoraat bij deze willen bedanken.

Verder zou ik het CODA, de faculteit Diergeneeskunde en met name Dr. X. Van Huffel, Ir. J.

Weerts en de mensen van de afdeling Contractueel Onderzoek van de Federale

Overheidsdienst van Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu willen

bedanken voor de nodige centjes (zeg maar €uro’s) voor dit en zelfs toekomstig onderzoek.

Of zoals Wim het elke vrolijke ochtend op StuBru zou uitdrukken: “Tim wordt betaald met

uw belastinggeld (en veel te veel dan nog ?!?)”

194 Dankwoord

Dan zijn er mijn vrienden. En van de zovele dingen die we samen meemaakten, zijn er tal van

momenten memorabel. Ze allemaal omschrijven zou wat te omslachtig worden (hoewel voor

sommigen gedacht wordt aan een pocketuitgave) en zelfs dan zouden woorden ongetwijfeld te

kort schieten. Bedankt Jurg voor het functioneren als lichtpuntje bij onbegrijpbare delen

tijdens examenperiodes of voor al de duistere en blijde momenten doorheen vele jaren; Pascal

voor de toffe avonden van de luidruchtige gesprekken met pindanootjes op café tot de stille

momenten (langs de Maas?!) waar enkel onze gedachtes elkaar nog kruisen; Jean voor de

steeds deftige, soms haast plechtige steun als ik situaties of ‘epithon’-woorden niet begrijp of

fout inschat; Sylvie voor haar taalcorrecties en de briefjes en mails die al jaren vol vreugde

worden ontvangen; Frank voor zijn hulp gaande van de grot-tot-berg filosofie tot aan mijn

carrièreplanning toe; Lipe voor de kleine (en ok, ook grote), maar altijd fijne momenten; Flor

voor al de tijd waarbij het leutig en plezant blijft, zelfs als het op ‘brokken maken’ neerkomt;

Frans voor de straightforward gesprekken en etentjes die hoewel ze soms ijskoud (tot

ongeveer -16°C) dreigen overkomen steeds met warmte worden gebracht; Sofie voor een

vriendin te zijn waarbij ik me steeds, zelfs met onuitgesproken woorden, begrepen voel; Mvo

voor telkens met me mee te gaan -letterlijk en figuurlijk- where no wheelchair went before; Jo

voor meer dan het ‘muzikale’ entertainment alleen; Wouter voor de huishoudelijke klusjes

(het mag eens gezegd); en al hun partners die (net als zij zelve) vrienden zijn geworden voor

het leven. Dan zijn er de vrienden die ik helaas te weinig zie of mail, maar bij wie ik ondanks

de andere paden die we veelal bewandelen (soms tot zelfs de gidsen ons niet kunnen volgen),

steeds kan aankloppen om me (met of zonder slaapzak in de hand) onmiddellijk thuis te

voelen: Els, Steve, Carina, Rits, Iels, Mark, Tom, Dreke, Bums, Danny, Leslie, Sabine, Swin,

Bien, Bert, Hannelore, Bart, de scouts en/of minivoetballers, en zovele anderen (die ik

mogelijks niet beloofde hun naam te vermelden)… het lijkt zo onmogelijk ze allemaal te

benoemen. Immers voor allemaal (reeds vermeld of in gedachte) geldt dat “best friends aren’t

those who hold the title ... best friends are those who hold our hearts”.

Ten laatste zou ik vooral mijn ouders, mijn zus Ines, mijn schoonbroer Patrick, en ook reeds

mijn neefje Joren en petekindje Mirre willen bedanken voor hun eeuwige steun en voor hun

liefde, die nooit wordt uitgesproken omdat stilte soms zo veel meer zegt.

Dankjewel !

Dankwoord 195

De jury:

Prof. Dr. Dr. h. c. A. de Kruif (voorzitter)

Faculteit Diergeneeskunde, UGent

Prof. Dr. F. Haesebrouck (promotor)


Prof. Dr. D. Maes (promotor)


Dr. P. Butaye (copromotor)

CODA/CERVA, Ukkel

Prof. Dr. M. Vaneechoutte (lees- en examencommissie)

Faculteit Geneeskunde, UGent

Prof. Dr. J. Mainil (lees- en examencommissie)

Faculteit Diergeneeskunde, Universiteit Luik

Prof. Dr. K. Houf (lees- en examencommissie)


Prof. Dr. L. Peelman (examencommissie)


Prof. Dr. H. Favoreel (examencommissie)


Dr. Ir. M. Baele (examencommissie)


197

CURRICULUM VITAE

ARTICLES

Stakenborg, T., J. Vicca, P. Butaye, H. Imberechts, J. Peeters, A. de Kruif, F. Haesebrouck, and D. Maes. 2005.

A multiplex PCR to identify porcine mycoplasmas present in broth cultures. Vet. Res. Comm. In Press.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, F.C. Minion, J. Peeters, A. de Kruif, and F. Haesebrouck. 2005.

Characterization of in vivo acquired resistance of Mycoplasma hyopneumoniae to macrolides and

lincosamides. Microb. Drug Resist. 11(3): 290-294.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, T. De Baere, R. Verhelst, J. Peeters, A. de Kruif, F. Haesebrouck,

and M. Vaneechoutte. 2005. Evaluation of amplified rDNA restriction analysis (ARDRA) for the

identification of Mycoplasma species. BMC Infect Dis. 5(1):46

Stakenborg, T., J. Vicca, R. Verhelst, P. Butaye, D. Maes, A. Naessens, G. Claeys, C. De Ganck, F.

Haesebrouck, and M. Vaneechoutte. 2005. Evaluation of tDNA-PCR for the identification of

Mollicutes. J. Clin. Microbiol. 43(9):4558-4566.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, J. Peeters, A. de Kruif, and F. Haesebrouck. 2005. The diversity

of Mycoplasma hyopneumoniae within and between herds using Pulsed-Field Gel Electrophoresis. Vet.

Microbiol. 109(1-2):29-36.

Vandekerchove, D. G. F., P. G. Kerr, A. P. Callebaut, H. J. Ball, T. Stakenborg, J. Mariën, and J. E. Peeters.

2002. Development of a capture ELISA for the detection of antibodies to enteropathogenic Escherichia

coli (EPEC) in rabbit flocks using intimin-specific monoclonal antibodies. Vet. Microbiol. 88(4):351-

366.

Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. Haesebrouck. 2003. Evaluation of

virulence of Mycoplasma hyopneumoniae field isolates. Vet. Microbiol. 97(3-4):177-190.

Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. Haesebrouck. 2004. In vitro

susceptibilities of Mycoplasma hyopneumoniae field isolates. Antimicrob. Agents Chemother.

48(11):4470-4472.

PATENT

Stakenborg, T., J. Mariën, D. Vandekerchove, and J. Peeters. Attenuated mutant enteropathogenic E. coli

(EPEC) strains, process for their production and their use. International Patent Application N°

PCT/EP00/05061, Filed 2/6/2000

198 Curriculum Vitae

ORAL PRESENTATIONS

Stakenborg, T., K. Carlens, D. Vandekerchove, J. Peeters. Adherence of enteropathogenic Escherichia coli to

intestinal villi in vitro. Microbial Adhesion and Virulence Meeting; Copenhagen, Denmark & Lund,

Sweden, 30 October-3 November 2000.

Stakenborg, T. Onderzoek naar een vaccin tegen enteropathogene E. coli bij vleeskonijnen. Laureate prize.

World Rabbit Association; Merelbeke, Belgium, 28th April 2004.

Stakenborg, T., D. Vandekerchove., L. Bohez., J. Mariën., and J. Peeters. The use of attenuated

enteropathogenic Escherichia coli (EPEC) as a vaccine. COST 848; Madrid, Spain, 24-26 June 2004.

Stakenborg, T., J. Vicca, P. Butaye., D. Maes, A. de Kruif, and F. Haesebrouck. 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, 7-12 July 2002.

CONFERENCE PROCEEDINGS

Stakenborg, T., D. Vandekerchove, K. Rülle, and J. Peeters. 2001. Protection des lapins vaccinés avec une

souche EPEC 3-/O15 délétée dans le gène eae contre une inoculation d'epreuve. World Rabbit Science.

9: 23-24. (Special issue concerning the 9ièmes Journées de la recherche Cunicole in Paris, France).

Bohez, L., T. Stakenborg, D. Vandekerchove, J. Peeters: Essai de protection des lapins vaccinés avec une

souche EPEC 2+/O132 délétéé dans le gène tir contre des inoculations d'épreuves hétérologues.

10ièmes Journées de la Recherche Cunicole; Paris, France, 19-20 November 2003.

Bohez, L., T. Stakenborg, H. Laevens, J. Peeters, and D. Vandekerchove: An attenuated 2+/O132∆tir

enteropathogenic Escherichia coli (EPEC) offers cross protection against a 3-/O15 challenge and partial

protection aganst an 8+/O103 challenge. 8ème Congrès Mondial de Cuniculture; Puebla, Mexico, 7-10

September 2004.

Bohez, L., T. Stakenborg, H. Laevens, J. Peeters, D. Vandekerchove: Different administration methods for the

3-/O15∆eae EPEC vaccine strain protecting meat rabbits against a 3-/O15 challenge: preliminary

results. 8ème Congrès Mondial de Cuniculture; Puebla, Mexico, 7-10 September 2004.

Bohez, L., L. Maertens, H. Laevens, T. Stakenborg, J. Peeters, and D. Vandekerchove: Use of a 3-/O15∆eae

enteropathogenic Escherichia coli vaccine in a rabbitry with mixed enteropathy problems: spreading

characteristics and protective effect. 8ème Congrès Mondial de Cuniculture; Puebla, Mexico, 7-10

September 2004.

Curriculum Vitae 199

REPORTS

Stakenborg, T., J. Vicca, S. Tistaert, P. Butaye, and H. Imberechts. 2002. Isolation, identification and diversity

of Mycoplasma hyopneumoniae strains. In Annual Report CODA. pp. 26-27.

Stakenborg, T., M. Vaneechoutte, F. Haesebrouck, and P. Butaye. 2004. Extending the application of tDNA-

PCR to identify mollicutes. In Scientific Report CODA. pp. 18-19.

Bohez, L., T. Stakenborg, J. Mariën, L. Maertens, D. Vandekerchove, and J. Peeters. 2004. Bescherming van

vleeskonijnen tegen colibacillaire diarree. FOD Volksgezondheid, Veiligheid van de Voedselketen en

Leefmilieu. Afdeling Contractueel Onderzoek. Synthesebrochure. pp. 1-108.

Bohez, L., T. Stakenborg, J. Mariën, L. Maertens, M. Van Hessche, D. Vandergheynst, J. Peeters, and D.

Vandekerchove. 2004. Development of live attenuated vaccine strains protecting meat rabbits against

enteropathogenic Escherichia coli (EPEC). In Scientific Report CODA. pp. 29-30.

POSTER PRESENTATIONS

Stakenborg, T., Rülle, K., Vandekerchove, D., Peeters, J: Intimin deletion mutants of rabbit enteropathogenic

Escherichia coli may be used as a vaccine against collibacillose. 6th International Veterinary

Immunology Congress, Uppsala, Sweden, 15-20 July 2001.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, A. de Kruif, and F. Haesebrouck. RAPD analysis of Belgian

Mycoplasma hyopneumoniae strains. 14th international congress of the International Organisation for

Mycoplasmology; Vienna, Austria, 7-12 July 2002.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, A. de Kruif, F. Haesebrouck. 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, 7-12 July 2002.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, A. de Kruif, and F. Haesebrouck, F. 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.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, A. de Kruif, and F. Haesebrouck. 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.

Stakenborg, T., J. Vicca, P. Butaye, D. Maes, A. de Kruif, and F. Haesebrouck. 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., J. Vicca, R. Verhelst, P. Butaye, D. Maes, A. Naessens, G. Clays, C. De Ganck, F.

Haesebrouck, and M. Vaneechoutte. Evaluation of tDNA-PCR for the identification of Mollicutes.

Belgian Society of Microbiology, Brussels, Belgium, 3rd December 2004.

200 Curriculum Vitae

Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, L. Devriese, and F. Haesebrouck.

Antibiotic susceptibility of Belgian Mycoplasma hyopneumoniae Field Isolates. 14th international

congress of the International Organisation for Mycoplasmology; Vienna, Austria, 7-12 July 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. 14th international congress of the

International Organisation for Mycoplasmology; Vienna, Austria, 7-12 July 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. 17th Congress of the International Pig

Veterinary Society; Ames, Iowa, USA, 2-5 June 2002.

Vicca, J., D. Maes, T. Stakenborg, P. Butaye, J. Peeters, A. de Kruif, F. Haesebrouck. Onderzoek naar

verschillen tussen Belgische Mycoplasma hyopneumoniae isolaten. IPVS Belgian branch; Merelbeke,

Belgium, 22nd November 2002.

Vicca, J, T. Stakenborg, D. Maes, P. Butaye, A. de Kruif, and F. Haesebrouck. Antimicrobial susceptibilities of

Mycoplasma hyopneumoniae field isolates. 11th annual meeting of the Flemish society for veterinary

epidemiology and economics; Torhout, Belgium, 11th December 2003.

Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. Haesebrouck. In vitro susceptibility

of Mycoplasma hyopneumoniae field isolates. 18th Congress of the International Pig Veterinary Society;

Hamburg, Germany, 27 June-1 July 2004.

identification of mollicutes and characterisation of...

Documents