veterinary

microbiology
Veterinary Microbiology 47 (1995) 183-190

In vitro amplification of the 16s rRNA genes from

Mycoplasma bovis and Mycoplasma agalactiae
by PCR
Yleana R. Chivez Gonz6lez a, Carlos Ros Bascufiana b,
G&an Biilske b, Jens G. Mattsson b, Carmen Fernbdez Molina lTa,
Karl-Erik Johansson bV*
aCentro National de Sanidad Agropecuaria, Apartado IO, San JosC de las Lajas, LA Habana, Cuba
b The National Veterinary Institute, Box 7073, S-750 07 Uppsala. Sweden

Received 5 December 1994; accepted 9 March 1995

Abstract

Mycoplasma bovis and Mycoplasma agalactiae are two very closely related species which cause
mastitis in cows and goats, respectively. M. bovis can also cause arthritis and respiratory disease in
cattle. It has recently been shown that the 16s rRNA sequences differ only in 8 nucleotide positions
between the two species [J.G. Mattsson, B. Guss and K.-E. Johansson ( 1994) FEMS Microbial.
Lett., 115: 325-3281. These nucleotide differences are distributed over the molecule in such a way
that it is difficult to design specific identification systems, based on PCR only, for M. bouis and M.
aguluctiue. Two different PCR systems based on 16s rRNA sequence data have, however, been
designed for these two species. The forward primers were identical in the two systems and comple-
mentary to a segment of the evolutionarily variable region V2. The reverse primers were complemen-
tary to the variable region V6, in which there are. two nucleotide differences between M. bovis and
M. agalactiae. The size of the PCR products, generated with these primers, was 360 bp. Cross-
amplification was obtained with the two species in the heterologous PCR systems, but with approxi-
mately a lOO-fold lower efficiency. Cross-amplification was not obtained with any other bovine or
caprine mycoplasma except for Mycoplasma sp. strain Al343 of the caprine group 7. The detection
limit of the PCR system for M. bouis with a reference culture was 4 X lo2 CFU/ml and of the PCR
system for M. agalactiae 2X 10’ CFu/ml. The M. bouis-PCR system was used to analyze nasal
samples of calves from a herd where an outbreak of pneumonia had occured and it proved possible
to detect M. bouis in these samples.

Keywords: Cattle; Contagious agalactia; Diagnostics; Goat; Mastitis; Mycoplasma agolactiae; Mycoplasma boois;
PCR; Respiratory disease

0378-l 135/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

ss010378-1135(95)00058-5
184 Y. Chavez Gonzdlez et al. / Veterinary Microbiology 47 (1995) 183-190

1. Introduction

Mycoplasma is the trivial name for all genera of a group of bacteria which lacks the cell
wall and has a low G + C content in the genome. About 125 species of mycoplasmas have
been described and they are currently classified in 8 genera (Tully et al., 1993). Many
mycoplasma species are pathogenic for animals, man and plants and they are, therefore, of
great concern in human and veterinary medicine as well as in plant pathology (Maniloff et
al., 1992; Ross, 1993). Mycoplasmas are often host specific and cattle (Gourlay and
Howard, 1979) and goats (Cottew, 1979) harbour a number of different species. Mycu-
plasma bovis is one of the important mycoplasmas that infect cattle and causes mastitis,
arthritis and respiratory disease (Boughton, 1979; Ter Laak et al., 1992). Mycoplusma
ugalactiue is the etiological agent of contagious agalactia in goats and sheep (Lambert,
1987; DaMassa et al., 1992) and this important species is closely related to M. Louis as
judged from biochemical and immunological methods ( Askaa and Em& 1976). Further-
more, the close relationship between M. bovis and M. agahctiue is also supported by the
fact that there are only eight nucleotide differences in the 16s rRNA sequences (Mattsson
et al., 1994).
Microbiological culture and immunological techniques are generally used for the detec-
tion and identification of hf. bovis and M. agalactiae. The former technique is, however,
time consuming and the latter is, in some cases, not sensitive enough. Hybridization with a
randomly cloned DNA fragment has been used for detection of M. bovis, but this system
was found to cross-hybridize with Mycoplusma arginini (McCully and Brock, 1992).
Another randomly cloned restriction fragment has been used as probe for detection of M.
agalactiue (Tola et al., 1994). Hybridisation with oligonucleotide probes complementary
to the 16s rRNA genes has also been used for detection and identification of M. bouis and
M. ugaluctiue (Mattsson et al., 1991). The sensitivity was assumed to be adequate for
certain diagnostic applications, because of the high copy number of the rRNA in the cell.
However, animals with subclinical or chronic disease only harbour a small number of
organisms, and samples from such animals probably have to be enriched in growth medium
before using the probe technique ( Mattsson et al., 199 1) _Detection of the organism by the
PCR technology is the method of choice when high sensitivity is needed (Saiki et al., 1988).
The purpose of this study was to develop two PCR assays based on in vitro amplification
of the 16s rRNA genes for detection of M. bovis and M. agalactiae and to use the M. bovis-
PCR system for analysis of nasal swab samples from calves suffering from respiratory
disease.

2. Materials and methods

2.1. Mycoplasma species and strains

The different mycoplasmas and mycoplasma strains used in this work are listed in
Table 1. The mycoplasmas were cultured in the appropriate growth medium (Bolske,

* Tel.: +46 18 67 40 00. Fax: +46 18 30 9162. E-mail: Karl-Erik.Johansson@sva.se

’ Present address: Institute de Medicina Tropical Pedro Kouri, Apartado 11500, La Habana, Cuba.
 Y. Chdvez Gonzdlez et al. / Veterinary Microbiology 47 (I 995) 183-l 90 185

Table 1
Acholeplasma and Mycoplasma species and strains used for determination of the specificities of the PCR systems
for Mycoplasma bovis ( Mbo-PCR) and Mycopiasma agalactiae (Mag-PCR)

Species Strain Main host Mbo-PCR Mag-PCR Source

Acholeplasma laidlawii PGgT Cattle _c nd‘ FAOIWHO”

Mycoplasma agalactiae PG2T Sheep, goat + +d +++ FAOIWHO
M. arginini G230’ Sheep, goat FAOI WHO
M. bovoculi Ml50183 Cattle nd SVACCh
M. bovigenitalium PG11* Cattle FAOIWHO
Ml93178 Cattle nd SVACC
M28/79C Cattle nd SVACC
M80/82 Cattle nd SVACC
Mycoplasma sp., bovine serogroup 7 PG50T Cattle FAOIWHO
M. bovirhinis PG43T Cattle nd FAOIWHO
M213184 Cattle nd SVACC
M359/87 Cattle nd SVACC
M. bovis DonettaT Cattle + + +e ++ FAOI WHO
M. califomicum ST-6T Cattle FAOI WHO
M. canadense M5/86 Cattle nd SVACC
M. capricolum subsp. capricolum Calif. kidT Goat nd FAOlWHO
M. capricolum subsp. capripneumoniae F3gT Goat nd FAOI WHO
M. dispar M26l79 Canle nd SVACC
M. fermentans PG18’ Man nd FAOIWHO
M. mycoides subsp. mycoides (LC”) Y-goatT Goat nd FAO/WHO
M. mycoides subsp. capri PG3T Goat nd FAOIWHO
M. mycoides subsp. mycoides ( SCb) PGIT Cattle nd FAOIWHO
M. putrefaciens KS-IT Goat nd FAOIWHO
Mycoplasma sp., caprine group 5 Goat 145 Goat nd FAOIWHO
Mycoplasma sp., caprine group 7 Al343 Goat +++ FAO/ WHO
Mycoplasma sp., caprine group 11 2D Goat nd FAO/WHO

“Large colony type. %mall colony type. ‘No amplification. %trong amplification. ‘Very strong amplification. ‘Not
done. gFAO/WHO Collaborating Center for Animal Mycoplasmas. hThe culture collection of the National Vet-
erinary Institute.

1988). The suspension cultures were then aliquoted and stored at - 70°C for later use in
the PCR experiments. The number of mycoplasmas were determined in the reference
cultures of M. bock and M. agalactiae as number of CFU/ml (Rodwell and Whitcomb,
1983).

2.2. Sample preparation

One ml of outgrown suspension cultures were centrifuged for 10 min at 12 000 g. The
pellet was washed once in 1 ml of phosphate buffered saline (PBS) by resuspension and
centrifugation. The pellet was then suspended in 1 ml of H20, heated in a boiling water
bath for 5 min to break the cell membranes, rapidly chilled on ice and stored at - 20°C until
use.
Fifteen nasal swab samples were collected from a Swedish herd of calves, previously
affected by an outbreak of respiratory disease due to M. bovis. The swabs were immersed
186 Y. Chavez Gonzrilez et al. /Veterinary Microbiology 47 (1995) 183-190

in 3 ml of transport medium consisting of 20% horse serum and 0.5 mg/ml of ampicillin
in PBS and sent to the laboratory. The time for transportation never exceeded 24 h. Myco-
plasma culture was performed by inoculation with 0.01 ml of the transport medium directly
to agar plates and with 0.3 ml to broth tubes, which were then subcultured to agar plates
(Bolske, 1988). M. bouis colonies were identified on the agar plates by indirect immuno-
fluorescence (Rosendal and Black, 1972). The swab samples were stored at - 20°C until
the PCR experiments were performed. One ml of the swab sample was centrifuged at 12 000
g for 10 min and the pellet was resuspended in 100 ~1 of HzO. The sample was then heated
at 100°C for 5 min and immediately chilled on ice before analysis by PCR.

2.3. PCR systems

The sequences of the 165 rRNA from M. agalactiae and M. bovis have been published
(Weisburg et al., 1989; Mattsson et al., 1994, respectively) and were obtained from
GenBank by the accession numbers M24290 and U02968, respectively. The sequences were
analyzed by using the sequence analysis software package from the Genetics Computer
Group (University of Wisconsin, USA) (Devereux et al., 1984). Target regions for the
PCR primers were selected and oligonucleotides complementary to these target regions
were synthesized with the DNA-synthesizer PCR Mate, model 39 1, from Applied Biosys-
terns (Foster City, Calif, USA). The sequences of the primers are given in Table 2.
In vitro amplification by PCR for the detection of M. bovis and M. agalactiae was
performed with 5 ~1 of the sample in a reaction mixture of 50 ~1 containing 1.5 mM MgCl,,
50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100,50 PM dNTP, 50 pmol of
each primer and 1 U of Taq DNA polymerase from Promega Co (Madison, Wis, USA).
The thermal profiles were as follows: Denaturation at 94°C for 45 s, primer annealing at
60°C for 1 min and extension at 72°C for 2 min. Hot start was used to avoid initial nonspecific
primer annealing (Chou et al., 1992). The amplifications were performed for 30 or 35
cycles in a model 480 thermocycler (Perkin-Elmer Cetus, Norwalk, Conn, USA) with a
final extension step at 72°C for 3 min. The PCR products were analyzed by agarose gel
( 1.5%) electrophoresis and visualized by staining with ethidium bromide. A mixture of
BglI and HimI cleaved pBR328 DNA from Boehringer Mannheim Biochemica (Mannheim,
FRG) was used as molecular size marker.
Table 2
Nucleotide sequences of the F’CR primers used for detection of Mycoplasma bovis and Mycoplasma agalactiae
by in vitro amplification

Species Designation Sequence

M. bovis MboF 5’.CCT T-l-T AGA -ITG GGA TAG CGG ATG-3’
MboR 5’.CCG TCA AGG TAG CAT CAT ‘I-K CTA T-3’
M. agalactiae MagF 5’.CCT TIT AGA 7-I-G GGA TAG CGG ATG-3’
MagR 5’.CCG TCA AGG TAG CGT CAT ‘MC CTA C-3’

The two nucleotides which are different in the reverse primers (MboR and MagR) for the two PCR system are
indicated in boldface. The 3’-end of the forward primers corresponds to nucleotide position 146 in the M. bovis
16s rRNA sequence according to the GenBank numbering and the 3’-end of the reverse primers corresponds to
position 458.
 Y. Ctivez Gonzdlez et al. /Veterinary Microbiology 47 (1995) 183-190 187

3. Results

3.1. Design of the PCR systems

One PCR system was designed for M. bovis (Mbo-PCR) and another system for M.
agalactiae (Mug-PCR) . The forward primers were identical in the two systems and com-
plementary to the evolutionarily variable region V2 as defined by Gray et al. ( 1984). The
reverse primers were complementary to a segment of the variable region V6 which has two
nucleotide differences between M. bovis and M. agaluctiae. The target region was selected
in such a way that one of these nucleotide differences was complementary to the 3’-end of
the reverse primer (Table 2). The theoretical size of the PCR products is 360 bp for both
M. bovis and M. agalactiae which was also experimentally verified (Fig. 1 and Fig. 2).

3.2. Specificity and sensitivity

All bovine mycoplasmas listed in Table 1 were analyzed with the Mbo-PCR system and
all caprine mycoplasmas were analyzed with the Mug-PCR system. Some of the bovine
mycoplasmas were also analyzed with the Mug-PCR system and vice versa. M. fermentuns
which is a human mycoplasma was analyzed with one of the systems, because it is closely
related to M. bovis and M. agaluctiae as judged from sequence analysis of 16s rRNA
( Weisburg et al., 1989). Cross-amplification was obtained when M. bovis and M. agalactiae
were analyzed with the heterologous PCR system, but with an efficiency which was approx-
imately lOO-fold lower as compared to the homologous PCR systems. Cross-amplification
was not obtained with any of the other closely related bovine and caprine mycoplasmas
tested or with M. fermentans, except for Mycoplasma sp. strain Al343 of the caprine group
7, which was strongly positive in both systems (Fig. 1 and Table 1) .
The sensitivities of the two PCR systems were determined with reference cultures of M.
bovis and M. agaluctiae with known CFU. The detection limit for the M. bovis-PCR system

< --- 360 bp

Fig. 1. Analysis of reference cultures of some caprine and bovine mycoplasmas by the Mycoplasma agalacriae-
PCR system. Lane 1, molecular size markers; lane 2-3, M. agalactiae; lane 4-5, M. mycoides subsp. mycoides
(SC); lane 6-7, M. arginini; lane 8-9, M. putrefaciens, lane 10-l 1, Mycoplasma sp., caprine group 5; lane 12-
13, Mycoplasma sp., caprine group 7; lane 14-15, Mycoplasma sp., caprine group 11; lane 16, negative control.
The mycoplasma samples were analyzed by FCR at two dilutions (corresponding to 5 ~1 and 0.5 ~1 of sample)
applied in the first and the second sample well, respectively.
188 Y. Chhez Gontilez et al. /Veterinary Microbiology 47 (1995) 183-190

12 3 4 5 6 7 8 9101112

<___ 360 bp

Fig. 2. Analysis of nasal swab samples from calves in a herd with respiratory disease by the Mycopkzsma bouis-
PCR system. Lane 1, molecular size markers; lane 2, negative control; lane 3, M321; lane 4, M33 1; lane 5, M341;
lane 6, M342; lane 7, M349, lane 8, M357; lane 9, M372; lane 10. M382; lane 11, positive control (M. bouis);
lane 12, negative control. The samples were analyzed after PCR at one dilution only.

with a reference culture was 4X 10’ CFU/ml and it was 2X 10’ CFU/ml for the M.
agalactiue-PCR system. The detection limits for the two species in the respective heterol-
ogous PCR system were about lOO-fold higher, due to the two mismatched base pairs in
the reverse primers.

3.3. Analysis of clinical samples

M. bovis was isolated from seven of the 15 nasal swab samples collected from calves of
a Swedish herd where an outbreak of respiratory disease had occured. In five of these
samples, h4. bovis was only detected on agar plates where the samples were subcultured
from broth and not on the direct plates. This observation indicated that there were less than
100 CFU of M. bovis organisms per ml in these samples. Six of the seven culture positive
samples were also positive in the M. bovis-F’CR system. The result of a PCR experiment of
some of these samples with the h4. bovis-specific system is shown in Fig. 2. The samples
M321, M341 and M342 were positive in PCR and also shown to contain M. bovis by
microbiological culture and immunofluorescence. The other samples were negative in PCR
and M. bouis could not be isolated from them.

4. Discussion

Cross-amplification was obtained with M. bovis and M. agalactiae in the heterologous

PCR system because these two species are closely related and have only eight nucleotide
differences in the 16s rRNA sequences which corresponds to 99.5% nucleotide similarity.
Two mismatched base pairs between one of the primers and its heterologous target was not
sufficient for absolute specificity, although one of them is in the 3’-end of the primer.
 Y. Chdvez Gonzdlez et al. /Veterinary Microbiology 47 (1995) 183-190 189

It is not surprising that Mycoplasma sp. strain A1343, which belongs to the caprine group
7, is amplified with both the Mbo-PCR and the Mug-PCR systems, since this strain is very
closely related to M. boois. This strain has, for instance, the same biochemical reactions as
M. bouis. Furthermore, protein mapping by two-dimensional polyacrylamide gel electro-
phoresis has shown that strain Al343 is very similar to M. bouis (Rodwell, 1982). It has
in fact been pointed out in 1982 by the Working Team for Caprine and Ovine Mycoplasmas
of the International Organization for Mycoplasmology that strain Al343 probably should
be classified as a variant strain of M. bovis and this is in agreement with our results.
The results from this study clearly demonstrated that the PCR can be used for detection
of M. bouis in nasal swabs from infected animals. The PCR technique is much faster than
conventional microbiological techniques for isolation as well as identification and results
can be obtained within 5 h. The sensitivities of both PCR systems, described in this work,
are adequate for most diagnostic purposes and the systems can probably also be used to
detect mycoplasmas in animals with subclinic or chronic disease. M. agalactiae has to the
knowledge of the authors never been isolated from cattle and M. bovis only occasionally
from goats ( DaMassa et al., 1992). These species can, therefore, in practice be regarded as
host-specific and the cross-amplifications with the two PCR systems are not critical in
diagnostic applications.
PCR systems for M. bovis (Hotzel et al., 1993) and M. agalactiae (Dedieu et al., 1994)
have recently been described. These PCR systems were based on sequences of randomly
cloned segments of the genome. The PCR systems described in the present work are useful
alternatives to the other PCR systems. The new PCR systems have the potential of being
extremely sensitive, since they are based on rRNA genes and can easily be modified for
direct amplification of the corresponding segment from the 16s rRNA molecule (van
Kuppeveld et al., 1992) which exists in high copy number in the cell.

Acknowledgements

We thank Eva Olsson for constructive criticism of the manuscript and Katrin Bergstrom,
Marianne Persson, Elisabeth Wilhelmsson and Stefan Jemstedt for valuable technical assis-
tance. This work was financially supported by the Swedish Agency for Research Cooper-
ation with Developing Countries (SAREC) .

References

Askaa, G. and Em@, H., 1976. Elevation of Mycoplasma agalactiae subsp. bouis to species rank: Mycoplasma
bouis (Hale et al.) comb. nov. Int. J. Syst. Bacterial., 26: 323-325.
Bolske, G., 1988. Survey of mycoplasma infections in cell cultures and a comparison of detection methods.
Zentralbl. Bakteriol. Hyg. A269: 331-340.
Boughton, E., 1979. Mycoplasma bouis mastitis. Vet. Bull., 49: 377-378.
Chou, Q., Russell, M., Birch, D.E., Raymond, J. and Bloch, W., 1992. Prevention of pre-PCR mis-priming and
primer dimerization improves low-copy-number amplifications. Nucleic Acids Res., 20: 1717-1723.
Cottew, G.S., 1979. Caprine-ovine mycoplasmas. In: J.G. Tully and R. F Whitcomb (Eds.), The Mycoplasmas.
Vol. II, Human and Animal Mycoplasmas. Academic Press, New York, pp 103-132.
190 Y. Chrivez Gonzcilez et al. /Veterinary Microbiology 47 (1995) 183-190

DaMassa, A.J., Wakenell, P.S. and Brooks, D.L., 1992. Mycoplasmas of goats and sheep. J. Vet. Diagn. Invest.,
4: 101-I 13.
Dedieu, L., Mady, V. and Lefevre, P.-C., 1994. Development of two polymerase chain reaction assays for the
identification of contagious agalactia related mycoplasmas. IOM Lett., 3: 31-32.
Devereux, J., Haeberli, P. and Smithies, 0.. 1984. A comprehensive set of sequence analysis programs for the
VAX Nucleic Acids Res., 12: 387-395.
Gourlay, R.N. and Howard, C.J., 1979. Bovine mycoplasmas, In: J.G. Tully and R.F. Whitcomb (Eds.), The
Mycoplasmas. Vol. II, Human and Animal Mycoplasmas. Academic Press, New York, pp 49-102.
Gray, M.W., Sankoff, D. and Cedergren, R.J., 1984. On the evolutionary descent of organisms and organelles: a
global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA. Nucleic Acids
Res., 12: 5837-5852.
Hotzel, H., Demuth, B., Sachse, K., Pllitsch, A. and PRitzner, H., 1993. Detection of Mycoplasma bouis using in
vitro deoxyribonucleic acid amplification. Rev. Sci. Tech. OIE 12: 58 l-59 I.
Lambert, M., 1987. Contagious agalactia of sheep and goats. Rev. Sci. Tech. OIE 6: 699-711.
McCully, M.A. and Brock, K.V., 1992. Development of a DNA hybridization probe for detection of Mycoplasma
bouis. J. Vet. Diagn. Invest., 4: 464-467.
Maniloff, J., McElhaney, R.N., Finch, L.R. and Baseman. J.B. (Eds.). 1992. Mycoplasmas: Molecular Biology
and Pathogenesis. American Society for Microbiology, Washington, DC.
Mat&son, J.G., Gersdorf, H., Gobel, U.B. and Johansson, K.-E., 1991. Detection of Mycoplasma boois and
Mycoplasma agdactiae by oligonucleotide probes complementary to 16s rRNA. Mol. Cell. Probes 5: 27-35.
Mattsson, J.G., Guss, B. and Johansson, K.-E., 1994. The phylogeny of Mycoplasma bouis as determined by
sequence analysis of the 16s rRNA gene. FEMS Microbial. Lett., 115: 325-328.
Rodwell, A.W., 1982. The protein fingerprints of mycoplasmas. Rev. Infect. Dis., 4: 8-17.
Rodwell, A.W. and Whitcomb, R.F., 1983. Methods for direct and indirect measurement of mycoplasma growth.
In: S. Razin and J.G. Tully (Eds.), Methods in Mycoplasmology. Vol. 1, Mycoplasma Characterization.
Academic Press, New York, pp 185-196.
Rosendal, S. and Black, F.T., 1972. Direct and indirect immunofluorescence of unfixed and fixed mycoplasma
colonies. Acta Pathol. Microbial. Stand. 80B: 615-622.
Ross, R.F., 1993. Mycoplasma - animal pathogens. In: I. Kahane and A. Adoni (Eds.), Rapid Diagnosis of
Mycoplasmas. Plenum Press, New York, pp 69-109.
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A., 1988.
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-
491.
Ter Laak, E.A., Wentink, G.H. and Zimmer, G.M., 1992. Increased prevalence of Mycoplasma bouis in the
Netherlands. Vet. Quart., 14: 100-104.
Tola, S., Rizzu, P. and Leori, G., 1994. A species-specific DNA probe for the detection of Mycoplasma agalacfiae.
Vet. Microbial., 41: 355-361.
Tully, J.G., BovC, J.M., La&ret, F. and Whitcomb, R.F., 1993. Revised taxonomy of the class Mollicutes: Proposed
elevation of a monophyletic cluster of arthropod-associated Mdllicutes to ordinal rank (Entotnoplasmatales
ord. nav.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasma-
raceae fam. nov.) from helical species (Spiroplasmataceae), and amended descriptions of the order Myco-
plasmatales, family Mycoplasmataceae. Int. J. Syst. Bacterial., 43: 378-385.
van Kuppeveld, F.J.M.. van der Logt, J.T.M., Angulo, A.F., van Zoest, M.J., Quint, W.G.V., Niestem, H.G.M.,
Galama, J.M.D. and Melchers, W.J.G., 1992. Genus-and species-specific identification of mycoplasmas by
16s rRNA amplification. Appl. Environ. Microbial., 58: 2606-2615.
Weisburg, W.G., Tully, J.G., Rose, D.L., Petzel, J.P., Oyaizu, H., Yang, D., Mandelco, L., Sechrest, J., Lawrence,
T.G., van Etten, J., Maniloff, J. and Woese, C.R., 1989. A phylogenetic analysis of the mycoplasmas: basis
for their classification. J. Bacterial., 171: 6455-6467.