The

The Veterinary Journal 168 (2004) 100–102

Veterinary Journalwww.elsevier.com/locate/tvjl

Short communication

Conservation of the uvrC gene sequence in Mycoplasma bovisand its use in routine PCR diagnosis

Anne Thomas a,*, Isabelle Dizier a, Annick Linden a, Jacques Mainil a,Joachim Frey b, Edy M. Vilei b

a Department of Bacteriology and Pathology, Faculty of Veterinary Medicine, University of Li�ege, B43A, Sart Tilman, Li�ege B-4000, Belgiumb Institute of Veterinary Bacteriology, University of Bern, L€anggass-Strasse 122, Postfach, Bern 3001, Switzerland

Accepted 7 October 2003

Keywords: Mycoplasma bovis; Polymerase chain reaction; Routine diagnosis; uvrC gene; Conservation

Mycoplasma bovis is a major cause of pneumonia and

arthritis in calves, and of mastitis and genital infections

in adult cows. It is responsible for high economic loss infeedlot cattle although it is often underestimated

(Nicholas et al., 2002) and is widely spread within the

bovine population in enzootically infected areas (Nich-

olas and Ayling, 2003).

As clinical and pathological signs are not character-

istic for M. bovis infection, laboratory diagnosis is nec-

essary. To detect M. bovis in clinical specimens, culture

is frequently used in combination with a sandwichELISA (Ball et al., 1994), or PCR-based detection and

identification. Initially, PCR tests were developed based

on the 16S rRNA genes and without further analysis

these assays failed to differentiate M. bovis from My-

coplasma agalactiae (Ch�avez Gonz�alez et al., 1995).

Moreover, the high degree of variation in the 16S rRNA

genes suggested the potential for misdiagnosis of closely

related Mycoplasma species using 16S rRNA-basedPCR assays (K€onigsson et al., 2002). Subramaniam and

colleagues (1998) developed a PCR based on the DNA

repair uvrC gene, which was shown to clearly differen-

tiate between M. bovis and M. agalactiae.

The present study aimed to analyze the polymor-

phisms that may occur in the uvrC gene among M. bovis

strains and to show the usefulness of the uvrC PCR test

adapted for the routine diagnosis of M. bovis. The PCRwas modified to reduce the volume of reagent required,

* Corresponding author. Tel.: +32-43664062/43664059; fax: +32-

43664122/43664565.

E-mail address: athomas@ulg.ac.be (A. Thomas).

1090-0233/$ - see front matter � 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tvjl.2003.10.006

with the aim to reduce costs and make it more suitable

for routine diagnostic laboratory use.

Ninety two strains of M. bovis isolated from variousorigins (summarized in Table 1) were confirmed by

sandwich ELISA (Ball et al., 1994). M. agalactiae strain

3990 (Pilo et al., 2003) was used as a negative control.

Type strains ofMycoplasma arginini,Mycoplasma dispar,

Mycoplasma alkalescens, Mycoplasma bovigenitalium,

Mycoplasma bovirhinis, Mycoplasma mycoides subsp.

mycoides small colony, Mycoplasma sp. bovine group 7,

and Acholeplasma laidlawii were also tested to evaluatethe specificity of the adapted PCR test. Aliquots of 200 lLof mycoplasma cultures that had been stored frozen at

)70 �C were grown in Friis or in modified Hayflick broth

medium (2mL) during 24–48 h at 37 �C (Ball et al., 1994).

Mycoplasmas (1 mL) were harvested by centrifugation at

8000g for 15 min, washed with PBS solution and re-

suspended in 150 lL sterile purified water. After boiling

(10 min) and centrifugation (12,000g for 1 min), 100 lLfrom the supernatant were collected. One microlitre of

each supernatant was added to 0.1 lL of Taq polymerase

(5U/lL,Roche), 0.4lLof 10lMprimerMBOUVRC2-L

(50-TTACGCAAGAGAATGCTTCA-30), 0.4 lL of 10

lMprimerMBOUVRC2-R (50-TAGGAAAGCACCCT

ATTGAT-30), 1 lL of dNTP mixture (containing 2 mM

of each dNTP), 1 lL of 10�MgCl2 buffer (100 mMTris–

HCl; 15 mMMgCl2; 500 mMKCl, pH 8.3) and 6.1 lL ofsterile purified water. The MBOUVRC PCR cycling was

performed as described by Subramaniamet al. (1998), i.e.,

reaction mixtures were subjected to 94 �C for 2 min, and

35 cycles of amplification with the parameters 30 s at

94 �C, 30 s at 52 �Cand 90 s at 72 �C. The PCR amplified a

Table 1

Mycoplasma bovis strains used in uvrC PCR

Country Origin of the

samples

Number of

strains

Years/period

of isolation

Belgium Lung 19 1990–2000

Nose 2 1990–2000

Milk 9 1980–2000

Passagedb 1 1990–2000

UKa Lung 40 1990–2000

Nose 3 1990–2000

Milk 4 1990–2000

Joint fluid 4 1990–2000

Pleural fluid 1 1990–2000

Pericardium 1 1990–2000

Passagedb 3 1990–2000

Germany Milkc 2 1980–1990

Nose 1 1990–2000

USA Milk 1d 1962

France Lung 1e Before 1999

aGreat Britain and Northern Ireland.b Passaged in Hayflick broth medium.cOne from an asymptomatic heifer.d Type strain.eRabbit strain.

Table 2

Mycoplasma bovis strains from which the uvrC gene was sequenced

Strains Years/period

of isolation

Country Origin of

the sample

PG45 1962 USA Milk

ML1 Before 1999 France Leprine lung

120/81 1980–1990 Germany Milk

221/89 1980–1990 Germany Asymptomatic

heifer (milk)

1897 1990–2000 Belgium Lung

39G 1990–2000 Belgium Lung

9585 1990–2000 Belgium Lung

9585P98 1990–2000 Belgium Passageda

2615 1990–2000 Belgium Lung

86p 1990–2000 Belgium Milk

2610 1990–2000 UK Joint fluid

2610P116 1990–2000 UK Passageda

2083 1990–2000 UK Joint fluid

2083P98 1990–2000 UK Passageda

2138 1990–2000 UK Milk

2138P98 1990–2000 UK Passageda

1783 1990–2000 UK Lung

2960 1990–2000 UK Lung

33B97 1990–2000 UK Lung

109B97 1990–2000 UK Lung

a Passaged in Hayflick broth medium.

A. Thomas et al. / The Veterinary Journal 168 (2004) 100–102 101

1626-bp fragment in all the 92M. bovis strains tested but

no amplification was observed inM. agalactiae and in the

other Mycoplasma species tested, underlining the speci-

ficity of the test. These 92 strains are a representative

population comprising various years of isolation, various

countries of origin, different pathological backgrounds

and different cultural features. Positive reactions were

obtained using only 10 lL of mix volume. This techniqueis reproducible and rapid and allows the identification of

M. bovis from small volumes of broth culture and in small

PCR mix volumes, in contrast to the protocol (mix vol-

umes of 50 lL) proposed by Subramaniam et al. (1998).

Sequences of the uvrC gene were obtained for 20 M.

bovis strains (Table 2) as follows: Mycoplasmas grown

24–48 h (1 mL) were harvested by centrifugation at 8000g

for 15 min, washed with phosphate-buffered saline (PBS)solution (140mMNaCl; 2.7mMKCl; 15mMKH2PO4; 8

mM Na2HPO4, pH 7.4) and re-suspended in 150 lLsterile purified water. Mycoplasma genomic DNA was

extracted by phenol/chloroform as described previously

(Su et al., 1990). DNA concentration was determined

spectrophotometrically with GeneQuantI (Amersham

Pharmacia Biotech). Approximately 2–5 ng of DNA was

submitted toMBOUVRC andMAGUVRCPCR tests ina 50 lL final volume as described by Subramaniam et al.

(1998).DNA sequencing of 2 lLof theMBOUVRCPCR

products (1626 bp) was performed in both directions

(sense and reverse) by using internal primers, with aDNA

Sequenator AB 3100 and the TaqDyeDeoxy Terminator

Cycle Sequencing Kit (Applied Biosystems) as described

by the manufacturer. The DNA sequence was assembled

using the Sequencher 3.0 software (GeneCodes). Align-

ments were done with PILEUP from the GCGWisconsin

package (Genetics Computer Group). Among the 20 M.

bovis strains tested, all but one (strain 2138P98) shared

100% identity with the sequence from type strain PG45

publishedbySubramaniamet al. (1998) under theEMBL/

GenBank Accession No. AF003959. Strain 2138P98

presented a single nucleotide change (G becoming A atposition 949of the 1716-bp coding sequence, resulting in a

change GGAGly to AGAArg). This strain originated from

bovine milk in the UK and had since been passaged 98

times. Theunpassaged strain sequencewas identical to the

type strain. A large number of M. bovis isolates used in

this study had shown different antigenic expression of the

vsps (Thomas et al., 2003). No difference in nucleotide

composition of uvrC was observed between strains orig-inating from a rabbit, cattle, different countries or differ-

ent isolation sites.

In summary, previous studies on the uvrC gene of M.

bovis showed the conservation of restriction patterns of

MBOUVRC PCR products when comparing the type

strain PG45 and 13 field isolates (Subramaniam et al.,

1998). By sequencing the uvrC gene of 20 strains and using

PCR to amplify 92 strains, this study has further con-firmed that the uvrC gene is a suitable conserved target for

diagnosis of M. bovis using PCR. Interestingly the one

strain that showed a single point polymorphism had been

adapted in the laboratory by passaging 98 times. As a

result of this study it is recommended that PCR of the

uvrC gene in M. bovis should be used for routine diag-

nosis. With the revised method it is low cost and ideal for

102 A. Thomas et al. / The Veterinary Journal 168 (2004) 100–102

confirming the identification of M. bovis. Despite the re-ported genetic variability in mycoplasmas, this study

confirms that the uvrC gene inM.bovis is stable and resists

normal mutation pressures. This underlines the useful-

ness of the uvrC PCR inM. bovis diagnosis with regard to

the sequence stability of this essential gene.

Acknowledgements

This study was possible thanks to the grant from the

BelgianMinist�ere de l�Agriculture (convention 6039). The

authors are grateful to Dr. Nicholas and Dr. Ayling

(Veterinary Laboratories Agency, Weybridge, Surrey,

UK), Dr. Ball (DARDNI, Belfast, UK), Dr. Poumarat

(AFSSA, Lyon, France), Dr. Sachse (BGVV, Jena, Ger-

many), Dr. Blanchard (INRA, Bordeaux, France), Dr.

Kempf (AFSSA, Ploufragan, France), Dr. Boucher (LesHerbiers, France), and T. Stakenborg (CODA-CERVA,

Brussels, Belgium) for providing some M. bovis strains.

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