Amplification of a 500-basepair fragment from cultured isolates of Mycobacterium bovis
The presence of a 500-bp fragment which amplifies a region from the genome of Mycobacterium bovis (J. G. Rodriguez, G. A. Meija, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131-2138, 1995) was evaluated by carrying out PCR on 121 M. bovis isolates. The M. bovis strains, previously characterized by culture and biochemical tests, were isolated from cattle in different regions of Argentina, Mexico, and Colombia. Four additional strains isolated from sea lions that belong to the M. tuberculosis complex were also included in the study. All of the isolates tested were PCR positive, rendering the expected 500-bp band and giving a correlation of 100% with previous microbiological characterization. Southern blot analysis revealed a common band of 1, 800 bp and a polymorphic high-molecular-mass hybridization pattern. The results show that this assay may be useful for diagnosis and identification of M. bovis in cattle.
JOURNAL OF CLINICAL MICROBIOLOGY,
July 1999, p. 2330–2332 Vol. 37, No. 7
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Ampliﬁcation of a 500-Base-Pair Fragment from Cultured
Isolates of Mycobacterium bovis
* JUAN CARLOS FISSANOTI,
PATRICIA DEL PORTILLO,
MANUEL ELKIN PATARROYO,
A ISABEL ROMANO,
AND ANGEL CATALDI
and Instituto de Inmunologı´a, Hospital San Juan de Dios,
Santafe´ de Bogota´,
D.C. Colombia, and Instituto de Biotecnologı´a CICV/INTA, Moro´n, Argentina
Received 25 November 1998/Returned for modiﬁcation 20 January 1999/Accepted 20 March 1999
The presence of a 500-bp fragment which ampliﬁes a region from the genome of Mycobacterium bovis (J. G.
Rodriguez, G. A. Meija, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131–2138, 1995)
was evaluated by carrying out PCR on 121 M. bovis isolates. The M. bovis strains, previously characterized by
culture and biochemical tests, were isolated from cattle in different regions of Argentina, Mexico, and Colom-
bia. Four additional strains isolated from sea lions that belong to the M. tuberculosis complex were also
included in the study. All of the isolates tested were PCR positive, rendering the expected 500-bp band and
giving a correlation of 100% with previous microbiological characterization. Southern blot analysis revealed a
common band of 1,800 bp and a polymorphic high-molecular-mass hybridization pattern. The results show
that this assay may be useful for diagnosis and identiﬁcation of M. bovis in cattle.
Bovine tuberculosis remains an important zoonosis in many
countries of the world. Cases of human tuberculosis of bovine
origin have increased in recent years (3, 5, 8, 19), and this
zoonosis has become a public health problem, as well as the
cause of signiﬁcant economic losses. Mycobacterium bovis, the
causative agent of bovine tuberculosis, infects both animals of
agricultural importance and wild mammals, which act as a
reservoir for the organism, making it difﬁcult to control the
The bovine tuberculin test is easy to perform on a large scale
on livestock, but it has the inconvenience of having a broad
range of speciﬁcity and sensitivity (13). Conﬁrmation of the
diagnosis is achieved by culture and biochemical assays. De-
spite the fact that microbiological culture is highly speciﬁc, a
positive result takes a long time to obtain and in most cases is
achieved after the animal has been sacriﬁced.
It is necessary to develop new diagnostic methods for bovine
tuberculosis which could identify M. bovis directly in biological
samples, such as milk or blood, without having to culture them
and which would also improve the predictive value of the
tuberculin test. Although the PCR has been successfully ap-
plied for the diagnosis of tuberculosis, routine application of a
PCR-based method requires that the target sequence be highly
speciﬁc for the microorganism and that it be present in all of
the strains isolated.
Rodrı´guez et al. (14) reported a PCR assay which ampliﬁes
a 500-bp fragment from the M. bovis genome by using the
JB21-JB22 primer pair. However, only a small number of iso-
lates were used in that study. The present work was performed
to determine whether this 500-bp fragment could be ampliﬁed
from the genome of different, previously characterized, M. bo-
Mycobacterial isolates and DNA extraction. A total of 121
isolates identiﬁed as M. bovis on the basis of growth in the
presence of pyruvate (scarce growth in glycerol), colony mor-
phology, and biochemical and enzymatic tests (niacin negative,
nitrate reduction negative, catalase negative, urease positive,
pyrazinamidase negative) were used in this study (9). Suscep-
tibility to thiophene-2-carboxylic acid hydrazide and p-amino-
salicylic acid was determined in egg solid medium with gluta-
mate added and without glycerol (9). In some cases, guinea
pigs and rabbits were inoculated in order to conﬁrm an M. bo-
vis identiﬁcation. M. tuberculosis H37Rv and M. bovis AN5
were used as reference strains. One hundred twelve of these
were bovine strains from Argentina (taken from six different
regions of the country), two were bovine strains from Mexico,
and seven were from Colombia. Four isolates belonging to the
M. tuberculosis complex were obtained from sea lions in Ar-
gentina and were also included in the study. Lymph nodes
(40%), lung tissue (45%), liver (10%), and samples from other
locations (5%) were collected during necropsy and cultured in
Stonebrink broth (16). All of them showed macroscopic lesions
typical of bovine tuberculosis. M. tuberculosis H37Rv, M. mi-
croti, M. africanum, and M. paratuberculosis were also analyzed
in the study. Chromosomal DNAs were isolated as described
by van Soolingen et al. (17), and 100 ng of each DNA was used
for PCR ampliﬁcation.
PCR assay. Primers JB21 and JB22 were synthesized on a
Pharmacia synthesizer. Primer sequences and performance of
the PCR were as reported previously by Rodriguez et al. (14).
The reactions were performed in a ﬁnal volume of 50 ml con-
taining 13 reaction buffer (Promega), 2.5 U of Taq polymerase
(Promega), 0.2 mM each deoxynucleoside triphosphate, 1.5
mM magnesium chloride, and 20 pmol of each primer. Target
DNA was denatured by incubation for 5 min at 94°C before
ampliﬁcation for 30 cycles of 94°C for 1 min, annealing at 68°C
for 1 min, and extension at 72°C for 1 min. All reactions
were carried out in an automated thermal cycler (Biometra).
After ampliﬁcation, 1/10 of the PCR mixture was analyzed by
gel electrophoresis in 1% agarose gels containing 0.5 mgof
ethidium bromide per ml.
Hybridization analysis. Genomic DNA was hydrolyzed with
the PvuII restriction enzyme (Gibco BRL). After separation
through agarose gel electrophoresis, the hydrolysate was trans-
ferred to a nylon membrane (Amersham) and hybridized with
the 500-bp ampliﬁed fragment labeled with [a-
random priming (Rediprime; Amersham).
* Corresponding author. Mailing address: Corporacio´n CorpoGen,
Calle 26 A No. 37-28, Santafe´ de Bogota´, D.C. Colombia. Phone:
57-1-368-5411. Fax: 57-1-3684987. E-mail: firstname.lastname@example.org.
Results. PCR was carried out on 121 M. bovis isolates by
using primers JB21 and JB22, which amplify a 500-bp fragment
of M. bovis (14). The 500-bp genomic fragment was present in
all of the M. bovis isolates used in this study, giving a 100% cor-
relation with the microbiological characterization. The frag-
ment was also ampliﬁed from the genome of the four M. tuber-
culosis complex strains isolated from sea lions. These isolates
were characterized as M. tuberculosis complex strains because
they shared molecular markers from both M. tuberculosis and
M. bovis (2, 15). No ampliﬁcation was observed for M. tuber-
culosis H37Rv, M. africanum, M. microti, and four M. paratuber-
To determine whether this 500-bp sequence is present as a
unique fragment in the M. bovis genome, Southern blot anal-
ysis was carried out by using 17 M. bovis isolates selected as
representatives of the total strains and the 500-bp fragment
was used as a hybridization probe. As shown in Fig. 1, the 500-
bp fragment hybridized to a 1.8-kb band present in all of the
samples tested, indicating a common location within the M. bo-
vis genome. In addition, positive signals were obtained with
one or two high-molecular-weight bands, between 7 and 10 kb.
Hybridization with these high-molecular-weight bands was poly-
morphic among the different isolates tested and also present
when M. tuberculosis genomic DNA was used (data not shown).
Discussion. The accurate diagnosis of bovine tuberculosis
remains, to this day, an elusive goal because no method has
been developed which can precisely detect the presence of the
microorganism in live animals. The tuberculin assay currently
used around the world renders highly variable results due to
problems with sensitivity and speciﬁcity. The tuberculin test
depends on several factors, including high-quality reagents, as
well as the immunological status of the animal. Furthermore, a
negative tuberculin test does not means that the animal is not
infected; on the other hand, a positive test can only mean a
delayed hypersensitivity reaction due to previous exposure. A
PCR-based assay, such as the one described here, could be used
to detect the presence of M. bovis in biological samples (such
as milk, blood, or nasal swabs) and thus become an important
tool for the control and eventual eradication of the disease.
A reliable PCR-based diagnostic assay must have a target
DNA sequence that is speciﬁc for the microorganism to be de-
tected and that must also be present in most, if not all, isolates
of the organism. The 500-bp fragment ampliﬁed by primers
JB21 and JB22 fulﬁlls the ﬁrst requirement, since it is ca-
pable of discriminating M. bovis from related strains, such as
M. avium, which is commonly isolated from cattle, and whose
tuberculin is used in the comparative intradermal tuberculin
test, and M. paratuberculosis, which is also pathogenic to cattle
(1). This fragment also fulﬁlls the second requirement: the
results of this study conﬁrm that the region ampliﬁed by prim-
ers JB21 and JB22 is conserved, since it was found in 121
isolates obtained from different geographic regions of Latin
America. It is also important to note that the M. tuberculosis
complex strains isolated from sea lions were PCR positive,
indicating that this sequence is also present in isolates from
other mammals, even thought they belong to a unique cluster
clearly different from M. bovis strains, and closely related to
M. tuberculosis (2, 4, 11, 15).
Initially, the fragment ampliﬁed by primers JB21 and JB22
was proposed by us to be exclusive to M. bovis and able to
discriminate between M. bovis and M. tuberculosis. However,
only 20 M. tuberculosis strains were included in that study (14).
As we included larger numbers of M. tuberculosis isolates, we
have observed that there are some strains which render a
500-bp ampliﬁcation band with the JB21-JB22 set of primers.
This, however, does not necessarily detract from the potential
beneﬁt of carrying out a PCR assay based on this sequence in
biological samples extracted from cattle, inasmuch as a positive
ampliﬁcation is indicative of the presence of an infectious
agent, be it human M. tuberculosis or M. bovis bovine tubercu-
FIG. 1. Southern blot analysis of different isolates of M. bovis. Genomic DNA was digested with restriction enzyme PvuII, and the 500-bp fragment of M. bovis was
used as a probe. Lanes 1 to 17 contain the following isolates of M. bovis: 1, 520; 2, 476; 3, 468; 4, 555; 5, 548; 6, 478; 7, T-372; 8, T-482; 9, 559; 10, 565; 11, 482; 12,
521; 13, 531; 14, 545; 15, 540; 16, 543; 17, 558. Lane 18 contains molecular weight markers. The values on the right are molecular weights in thousands.
VOL. 37, 1999 NOTES 2331
losis. Studies are currently being carried out using a large panel
of M. tuberculosis strains to determine the percentage of JB21-
JB22-positive ampliﬁcations. We are also using different
molecular markers, such as spoligotyping (10), oxyR (7), and
mtp40 (6), with the aim of determining whether these strains
might belong to a cluster of M. tuberculosis, similar to what is
occurring with the sea lion isolates.
PCR-based assay has been successfully used for the detec-
tion of M. tuberculosis. Recently, however, it has been shown
that some M. tuberculosis strains lack speciﬁc target sequences
such as IS6110 or mtp40 (11, 18). This could be due to the
presence of mutations or genomic rearrangements. So far,
all of the M. bovis strains tested by the assay described here
contain the 500-bp target sequence, indicating that this frag-
ment is conserved among M. bovis strains. Field test evaluation
of this PCR as a diagnostic tool for M. bovis detection is being
carried out by using milk and blood as biological samples in
order to demonstrate the validity of the test for the detection
of bovine tuberculosis.
1. Bauerfeind, R., S. Benazzi, R. Weiss, T. Schliesser, H. Willems, and G.
Baljer. 1996. Molecular characterization of Mycobacterium paratuberculosis
isolates from sheep, goats, and cattle by hybridization with a DNA probe to
insertion element IS900. J. Clin. Microbiol. 34:1617–1621.
2. Bernardelli, A., R. Bastida, P. Loureiro, C. Michelis, M. I. Romano, A.
Cataldi, and E. Costa. 1996. Tuberculosis in sea lions and fur seals from the
southwestern Atlantic Ocean. Rev. Sci. Tech. O. I. E. (Off. Int. Epizoot.) 15:
3. Brett, J. L., and M. W. Humble. 1991. Incidence of human tuberculosis
caused by Mycobacterium bovis. N. Z. Med. J. 104:13–14.
4. Cousins, D. V., B. R. Francis, B. L. Gow, D. M. Collins, C. H. McGlashan,
A. Gregory, and R. M. Mackenzie. 1990. Tuberculosis in captive seals: bac-
teriological studies on an isolate belonging to the Mycobacterium tuberculosis
complex. Res. Vet. Sci. 48:196–200.
5. de Kantor, I. N., and V. Ritacco. 1994. Bovine tuberculosis in Latin America
and the Caribbean: current status, control and eradication programs. Vet.
6. Del Portillo, P., L. A. Murillo, and M. E. Patarroyo. 1991. Ampliﬁcation of
a species-speciﬁc DNA fragment of Mycobacterium tuberculosis and its pos-
sible use in diagnosis. J. Clin. Microbiol. 29:2163–2168.
7. Espinosa de los Minteros, L. E., J. C. Galan, M. Gutierrez, S. Samper, J. F.
Garcia Marin, C. Martin, L. Dominguez, L. de Rafael, F. Baquero, E.
Gomez-Mampaso, and J. Blazquez. 1998. Allele-speciﬁc PCR method based
on pncA and oxyR sequences for distinguishing Mycobacterium bovis from
Mycobacterium tuberculosis: intraspeciﬁc M. bovis pncA sequence polymor-
phism. J. Clin. Microbiol. 36:239–242.
8. Fanning, A., and S. Edwards. 1991. Mycobacterium bovis infection in human
beings in contact with elk (Cervus elaphus) in Alberta, Canada. Lancet 338:
9. Grange, J. M., and M. D. Yates. 1994. Guidelines for speciation within the
Mycobacterium tuberculosis complex. WHO/Zoom./94.174. World Health
Organization Veterinary Public Health Unit, Geneva, Switzerland.
10. Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S.
Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van
Embden. 1997. Simultaneous detection and strain differentiation of Myco-
bacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35:
11. Liebana, E., A. Aranaz, B. Francis, and D. Cousins. 1996. Assessment of
genetic markers for species differentiation within the Mycobacterium tuber-
culosis complex. J. Clin. Microbiol. 34:933–938.
12. O’Reilly, L. M., and C. J. Daborn. 1995. The epidemiology of Mycobacterium
bovis infection in animals and man: a review. Tubercle Lung Dis. 76:1–46.
13. O’Reilly, L. M. 1992. Speciﬁcity and sensitivity of tuberculin test, p. 83–139.
In A. A. M. Moussa, O. Lotﬁ, S. Mahair, et al. (ed.), Proceedings of the
International Conference on Animal Tuberculosis in Africa and the Middle
East. General Organization for Veterinary Services, Cairo, Egypt.
14. Rodriguez, J. G., G. A. Mejia, P. Del Portillo, M. E. Patarroyo, and L. A.
Murillo. 1995. Species-speciﬁc identiﬁcation of Mycobacterium bovis by PCR.
15. Romano, M. I., A. Alito, F. Bigi, J. C. Fisanotti, and A. Cataldi. 1995. Genetic
characterization of mycobacteria from South American wild seals. Vet. Mi-
16. Stonebrink, B. 1958. The use of a pyruvate containing egg medium in the
culture of isoniazid resistant strains of Mycobacterium tuberculosis var.
Hominis. Acta Tuberc. Scand. 35:67–80.
17. Van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, D. R. Soll, and
J. D. A. van Embden. 1991. The occurrence and stability of insertion se-
quences in Mycobacterium tuberculosis complex strains: evaluation of IS-
dependent DNA polymorphism as a tool in the epidemiology of tuberculosis.
J. Clin. Microbiol. 29:2578–2586.
18. Weil, A., B. B. Plikaytis, W. R. Butler, C. L. Woodley, and T. M. Shinnick.
1996. The mtp40 gene is not present in all strains of Mycobacterium tuber-
culosis. J. Clin. Microbiol. 34:2309–2311.
19. World Health Organization. 1994. Zoonotic tuberculosis (Mycobacterium
bovis): memorandum from a WHO meeting (with the participation of FAO).
Bull. W. H. O. 72:851–857.
2332 NOTES J. CLIN.MICROBIOL.