identification of mollicutes and characterisation of...
TRANSCRIPT
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.
10 Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma
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.
Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma 11
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
12 Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma
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
Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma 13
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.
14 Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma
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
Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma 15
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
16 Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma
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,
Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma 17
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.
7. Bradbury, J. M. 2001. International committee on systematic bacteriology, subcommittee on the
taxonomy of Mollicutes. Int. J. Syst. Evol. Microbiol. 51:2227-2230.
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.
18 Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma
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.
Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma 19
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.
20 Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma
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.
Short introduction to the class of Mollicutes with emphasis on the genus Mycoplasma 21
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
24 Molecular techniques to detect, identify & type mycoplasmas
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
Molecular techniques to detect, identify & type mycoplasmas 25
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.
26 Molecular techniques to detect, identify & type mycoplasmas
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
Molecular techniques to detect, identify & type mycoplasmas 27
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).
28 Molecular techniques to detect, identify & type mycoplasmas
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
Molecular techniques to detect, identify & type mycoplasmas 29
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
30 Molecular techniques to detect, identify & type mycoplasmas
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
Molecular techniques to detect, identify & type mycoplasmas 31
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).
32 Molecular techniques to detect, identify & type mycoplasmas
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).
Molecular techniques to detect, identify & type mycoplasmas 33
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
34 Molecular techniques to detect, identify & type mycoplasmas
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).
Molecular techniques to detect, identify & type mycoplasmas 35
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.
36 Molecular techniques to detect, identify & type mycoplasmas
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).
Molecular techniques to detect, identify & type mycoplasmas 37
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.
38 Molecular techniques to detect, identify & type mycoplasmas
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
Molecular techniques to detect, identify & type mycoplasmas 39
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.
40 Molecular techniques to detect, identify & type mycoplasmas
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
Molecular techniques to detect, identify & type mycoplasmas 41
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).
42 Molecular techniques to detect, identify & type mycoplasmas
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
Molecular techniques to detect, identify & type mycoplasmas 43
(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
44 Molecular techniques to detect, identify & type mycoplasmas
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.
Molecular techniques to detect, identify & type mycoplasmas 45
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.
46 Molecular techniques to detect, identify & type mycoplasmas
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.
Molecular techniques to detect, identify & type mycoplasmas 47
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.
Microbiol. 42:746-752.
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.
22. Bradbury, J. M. 2001. International committee on systematic bacteriology, subcommittee on the
taxonomy of Mollicutes. Int. J. Syst. Evol. Microbiol. 51:2227-2230.
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.
Microbiol. 78:175-181.
48 Molecular techniques to detect, identify & type mycoplasmas
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.
Molecular techniques to detect, identify & type mycoplasmas 49
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 Molecular techniques to detect, identify & type mycoplasmas
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.
Molecular techniques to detect, identify & type mycoplasmas 51
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.
52 Molecular techniques to detect, identify & type mycoplasmas
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.
Clin. Microbiol. 41:5071-5079.
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.
Molecular techniques to detect, identify & type mycoplasmas 53
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.
Microbiol. 54:23-29.
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.
54 Molecular techniques to detect, identify & type mycoplasmas
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.
J. Clin. Microbiol. 42:2609-2617.
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.
J. Clin. Microbiol. 39:1882-1888.
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
Microbiol. 85:111-123.
Molecular techniques to detect, identify & type mycoplasmas 55
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.
56 Molecular techniques to detect, identify & type mycoplasmas
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.
Clin. Microbiol. 30:1499-1504.
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.
Molecular techniques to detect, identify & type mycoplasmas 57
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.
58 Molecular techniques to detect, identify & type mycoplasmas
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.
158. Rocha, E. P., and A. Blanchard. 2002. Genomic repeats, genome plasticity and the dynamics of
Mycoplasma evolution. Nucleic Acids Res. 30:2031-2042.
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.
Molecular techniques to detect, identify & type mycoplasmas 59
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.
60 Molecular techniques to detect, identify & type mycoplasmas
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.
Microbiol. 35:339-346.
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.
Molecular techniques to detect, identify & type mycoplasmas 61
189. van Belkum, A. 1999. The role of short sequence repeats in epidemiologic typing. Curr. Opin.
Microbiol. 2:306-311.
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.
62 Molecular techniques to detect, identify & type mycoplasmas
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.
Molecular techniques to detect, identify & type mycoplasmas 63
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.
64
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
68
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.
72 Evaluation of ARDRA for the identification of Mycoplasma species
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),
Evaluation of ARDRA for the identification of Mycoplasma species 73
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
74 Evaluation of ARDRA for the identification of Mycoplasma species
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.
Evaluation of ARDRA for the identification of Mycoplasma species 75
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.
76 Evaluation of ARDRA for the identification of Mycoplasma species
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.
Evaluation of ARDRA for the identification of Mycoplasma species 77
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.
78 Evaluation of ARDRA for the identification of Mycoplasma species
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).
Evaluation of ARDRA for the identification of Mycoplasma species 79
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.
80 Evaluation of ARDRA for the identification of Mycoplasma species
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
Evaluation of ARDRA for the identification of Mycoplasma species 81
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
82 Evaluation of ARDRA for the identification of Mycoplasma species
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
Evaluation of ARDRA for the identification of Mycoplasma species 83
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
)
84 Evaluation of ARDRA for the identification of Mycoplasma species
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)
Evaluation of ARDRA for the identification of Mycoplasma species 85
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)
86 Evaluation of ARDRA for the identification of Mycoplasma species
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)
Evaluation of ARDRA for the identification of Mycoplasma species 87
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
)
88 Evaluation of ARDRA for the identification of Mycoplasma species
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
)
Evaluation of ARDRA for the identification of Mycoplasma species 89
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
)
90 Evaluation of ARDRA for the identification of Mycoplasma species
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
)
Evaluation of ARDRA for the identification of Mycoplasma species 91
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)
92 Evaluation of ARDRA for the identification of Mycoplasma species
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)
Evaluation of ARDRA for the identification of Mycoplasma species 93
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
)
94 Evaluation of ARDRA for the identification of Mycoplasma species
Evaluation of ARDRA for the identification of Mycoplasma species 95
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
96 Evaluation of ARDRA for the identification of Mycoplasma species
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.
Evaluation of ARDRA for the identification of Mycoplasma species 97
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.
7. Bradbury, J. M. 2001. International committee on systematic bacteriology, subcommittee on the
taxonomy of Mollicutes. Int. J. Syst. Evol. Microbiol. 51:2227-2230.
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.
98 Evaluation of ARDRA for the identification of Mycoplasma species
12. 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.
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.
Evaluation of ARDRA for the identification of Mycoplasma species 99
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.
27. 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.
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.
31. 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.
32. 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.
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.
Clin. Microbiol. 34:149-158.
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.
100 Evaluation of ARDRA for the identification of Mycoplasma species
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
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 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.
Evaluation of tDNA-PCR for the identification of Mollicutes 103
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.
Materials and methods
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
104 Evaluation of tDNA-PCR for the identification of Mollicutes
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).
Evaluation of tDNA-PCR for the identification of Mollicutes 105
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)
106 Evaluation of tDNA-PCR for the identification of Mollicutes
Received as Final Identification tDNA-PCR based ID Isolated/ Date of Strain name Culture (Techniqueb) identification Received Isolation medium
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
Evaluation of tDNA-PCR for the identification of Mollicutes 107
Received as Final Identification tDNA-PCR based ID Isolated/ Date of Strain name Culture (Techniqueb) identification Received Isolation medium
fromc (mm/yy)
U. urealyticum NA U. urealyticum MYP088 VUB NA Serotype 04 A7
U. urealyticum NA U. urealyticum MYP089 VUB NA Serotype 05 A7
U. parvum NA U. parvum MYP090 VUB NA Serotype 06 A7
U. urealyticum NA U. urealyticum MYP091 VUB NA Serotype 07 A7
U. urealyticum NA No peaks MYP092 VUB NA Serotype 08 A7
U. urealyticum NA U. urealyticum MYP093 VUB NA Serotype 09 A7
U. urealyticum NA U. urealyticum MYP094 VUB NA Serotype 10 A7
U. urealyticum NA U. urealyticum MYP095 VUB NA Serotype 11 A7
U. urealyticum NA U. urealyticum MYP096 VUB NA Serotype 12 A7
U. urealyticum NA No peaks MYP097 VUB NA Serotype 13 A7
U. parvum NA U. parvum MYP098 VUB NA Serotype 14 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 No peaks MYP101 VUB 03/04 040330/8 A7
U. urealyticum NA No peaks MYP102 VUB 03/04 040330/7 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
M. hyorhinis M. hyorhinis (P) M. hyorhinis MYP005 CODA 08/00 MhyorF6A NHS20
M. hyorhinis M. hyorhinis (P) M. hyorhinis MYP006 CODA 01/01 MhyorF9A NHS20
108 Evaluation of tDNA-PCR for the identification of Mollicutes
Received as Final Identification tDNA-PCR based ID Isolated/ Date of Strain name Culture (Techniqueb) identification Received Isolation medium
fromc (mm/yy)
M. hyosynoviae M. hyosynoviae (A) M. hyosynoviae MYP059 DFVF NA Mp6 NA
M. hyosynoviae M. hyosynoviae (A) M. hyosynoviae MYP060 DFVF NA Mp96 NA
M. hyosynoviae M. hyosynoviae (A) M. hyosynoviae MYP061 DFVF NA Mp178 NA
M. hyosynoviae M. hyosynoviae (A) M. hyosynoviae MYP062 DFVF NA Mp356 NA
M. hyosynoviae M. hyosynoviae (A) M. hyosynoviae MYP063 DFVF NA Mp1023 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
Evaluation of tDNA-PCR for the identification of Mollicutes 109
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.
110 Evaluation of tDNA-PCR for the identification of Mollicutes
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
Evaluation of tDNA-PCR for the identification of Mollicutes 111
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
112 Evaluation of tDNA-PCR for the identification of Mollicutes
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
Evaluation of tDNA-PCR for the identification of Mollicutes 113
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.
114 Evaluation of tDNA-PCR for the identification of Mollicutes
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.
Evaluation of tDNA-PCR for the identification of Mollicutes 115
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.
116 Evaluation of tDNA-PCR for the identification of Mollicutes
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).
Evaluation of tDNA-PCR for the identification of Mollicutes 117
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
118 Evaluation of tDNA-PCR for the identification of Mollicutes
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.
Evaluation of tDNA-PCR for the identification of Mollicutes 119
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.
Clin. Microbiol. 39:1436-1442.
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.
8. Bradbury, J. M. 1997. International committee on systematic bacteriology, subcommittee on the
taxonomy of Mollicutes. Int. J. Syst. Evol. Microbiol. 47:911-914.
9. Bradbury, J. M. 2001. International committee on systematic bacteriology, subcommittee on the
taxonomy of Mollicutes. Int. J. Syst. Evol. Microbiol. 51:2227-2230.
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.
J. Clin. Microbiol. 39:162-169.
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.
16. 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.
120 Evaluation of tDNA-PCR for the identification of Mollicutes
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.
21. Kobisch, M., and N. F. Friis. 1996. Swine mycoplasmoses. Rev. Sci. Tech. 15:1569-1605.
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.
25. 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.
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.
Clin. Microbiol. 30:1499-504.
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.
Evaluation of tDNA-PCR for the identification of Mollicutes 121
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.
Microbiol. 3:613-625.
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.
122 Evaluation of tDNA-PCR for the identification of Mollicutes
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
1 Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium 2 Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke,
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,
A multiplex PCR to identify porcine mycoplasmas present in broth cultures 125
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.
Materials and methods
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
126 A multiplex PCR to identify porcine mycoplasmas present in broth cultures
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
A multiplex PCR to identify porcine mycoplasmas present in broth cultures 127
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.
128 A multiplex PCR to identify porcine mycoplasmas present in broth cultures
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
A multiplex PCR to identify porcine mycoplasmas present in broth cultures 129
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
130 A multiplex PCR to identify porcine mycoplasmas present in broth cultures
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
A multiplex PCR to identify porcine mycoplasmas present in broth cultures 131
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.
132 A multiplex PCR to identify porcine mycoplasmas present in broth cultures
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.
A multiplex PCR to identify porcine mycoplasmas present in broth cultures 133
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.
18. 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.
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.
21. Kobisch, M., and N. F. Friis. 1996. Swine mycoplasmoses. Rev. Sci. Tech. 15:1569-1605.
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.
Microbiol. 33:893-897.
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.
26. 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.
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.
134 A multiplex PCR to identify porcine mycoplasmas present in broth cultures
29. 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.
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
1 Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium 2 Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke,
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
Diversity of M. hyopneumoniae within and between herds using PFGE 137
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
138 Diversity of M. hyopneumoniae within and between herds using PFGE
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
Diversity of M. hyopneumoniae within and between herds using PFGE 139
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
140 Diversity of M. hyopneumoniae within and between herds using PFGE
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).
Diversity of M. hyopneumoniae within and between herds using PFGE 141
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.
142 Diversity of M. hyopneumoniae within and between herds using PFGE
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.
Diversity of M. hyopneumoniae within and between herds using PFGE 143
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
144 Diversity of M. hyopneumoniae within and between herds using PFGE
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.
Diversity of M. hyopneumoniae within and between herds using PFGE 145
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 Véronique Collet and Sara Tistaert for skilful technical assistance and
Annelies Pil for the numerous inspiring discussions.
References
1. 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.
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.
5. 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.
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.
8. Friis, N. F. 1975. Some recommendations concerning primary isolation of Mycoplasma suipneumoniae
and Mycoplasma flocculare a survey. Nord. Vet. Med. 27:337-339.
9. Goodwin, R. F. 1985. Apparent reinfection of enzootic-pneumonia-free pig herds: search for possible
causes. Vet. Rec. 116:690-694.
10. 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.
11. Hong, Y., and A. Chuah. 2003. A format for databasing and comparison of AFLP fingerprint profiles.
BMC Bioinformatics. 4:7.
146 Diversity of M. hyopneumoniae within and between herds using PFGE
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.
14. Kobisch, M., and N. F. Friis. 1996. Swine mycoplasmoses. Rev. Sci. Tech. 15:1569-1605.
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.
16. 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.
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.
21. 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.
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.
25. 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.
Diversity of M. hyopneumoniae within and between herds using PFGE 147
26. 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.
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.
32. 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.
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.
34. 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.
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.
148 Diversity of M. hyopneumoniae within and between herds using PFGE
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.
39. 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.
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
1 Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium 2 Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke,
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.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 151
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).
152 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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%.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 153
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).
154 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 155
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.
156 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 157
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).
158 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 159
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.
160 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 161
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
162 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 163
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
164 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 165
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
166 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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
Safety and Environment (Grant number S-6136).
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
1. 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.
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.
6. 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.
7. Done, S. H. 1991. Environmental factors affecting the severity of pneumonia in pigs. Vet. Rec.
128:582-586.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 167
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.
9. Friis, N. F. 1975. Some recommendations concerning primary isolation of Mycoplasma suipneumoniae
and Mycoplasma flocculare a survey. Nord. Vet. Med. 27:337-339.
10. Harasawa, R., K. Asada, and I. Kato. 1995. A novel repetitive sequence from Mycoplasma
hyopneumoniae. J. Vet. Med. Sci. 57:557-558.
11. 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.
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.
19. 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.
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.
168 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
21. 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.
22. 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.
23. 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.
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.
26. Rocha, E. P., and A. Blanchard. 2002. Genomic repeats, genome plasticity and the dynamics of
Mycoplasma evolution. Nucleic Acids Res. 30:2031-2042.
27. Rosengarten, R., and D. Yogev. 1996. Variant colony surface antigenic phenotypes within
mycoplasma strain populations: implications for species identification and strain standardization. J.
Clin. Microbiol. 34:149-158.
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.
31. Urwin, R., and M. C. Maiden. 2003. Multi-locus sequence typing: a tool for global epidemiology.
Trends Microbiol. 11:479-487.
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.
Comparison of molecular techniques for the typing of M. hyopneumoniae isolates 169
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.
35. 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.
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.
170 Comparison of molecular techniques for the typing of M. hyopneumoniae isolates
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.
174 General Discussion
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
General Discussion 175
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.
176 General Discussion
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
General Discussion 177
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
178 General Discussion
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).
General Discussion 179
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
180 General Discussion
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.
5. Bradbury, J. M. 2001. International committee on systematic bacteriology, subcommittee on the
taxonomy of Mollicutes. Int. J. Syst. Evol. Microbiol. 51:2227-2230.
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.
8. 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.
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.
General Discussion 181
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.
13. Friis, N. F. 1975. Some recommendations concerning primary isolation of Mycoplasma suipneumoniae
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.
16. 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.
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.
182 General Discussion
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.
27. 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.
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.
30. 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.
31. 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.
32. 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.
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.
General Discussion 183
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.
38. 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.
39. 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.
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.
41. Urwin, R., and M. C. Maiden. 2003. Multi-locus sequence typing: a tool for global epidemiology.
Trends Microbiol. 11:479-487.
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.
184
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.
188
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.
192
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)
Faculteit Diergeneeskunde, UGent
Prof. Dr. D. Maes (promotor)
Faculteit Diergeneeskunde, UGent
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)
Faculteit Diergeneeskunde, UGent
Prof. Dr. L. Peelman (examencommissie)
Faculteit Diergeneeskunde, UGent
Prof. Dr. H. Favoreel (examencommissie)
Faculteit Diergeneeskunde, UGent
Dr. Ir. M. Baele (examencommissie)
Faculteit Diergeneeskunde, UGent
196
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.