6255 Veterinary Microbiology 1542011152155
6255 Veterinary Microbiology 1542011152155
6255 Veterinary Microbiology 1542011152155
Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic
A R T I C L E I N F O A B S T R A C T
Article history: Rapid and specific identification of Brucella suis at the biovar level is necessary because
Received 18 February 2011 some of the biovars that infect animals are pathogenic for humans. None of the molecular
Received in revised form 23 June 2011 typing methods described so far are able to discriminate B. suis biovars in a single test and
Accepted 28 June 2011
differentiation of B. suis from Brucella canis by molecular approaches can be difficult. This
article describes a new multiplex PCR assay, Suis-ladder, for fast and accurate
Keywords:
identification of B. suis at the biovar level and the differentiation of B. suis, B. canis and
Brucella suis
Brucella microti. An advancement of the original Bruce-ladder PCR protocol which allows
Brucella canis
Bruce-ladder the correct discrimination of all known Brucella species is also described.
PCR ß 2011 Elsevier B.V. All rights reserved.
2.1. Strains
* Corresponding author at: Departamento de Microbiologı́a y Para-
sitologı́a, Universidad de Navarra, c/ Irunlarrea n8 1, 31008, Pamplona,
Spain. Tel.: +34 948 425600; fax: +34 948 425649. To ensure an adequate diversity, a representative
E-mail address: ilgoni@unav.es (I. López-Goñi). collection of B. suis and B. canis reference strains and field
0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2011.06.035
I. López-Goñi et al. / Veterinary Microbiology 154 (2011) 152–155 153
Table 1
Oligonucleotides used in the Suis-ladder multiplex PCR assay.
BMEI1426 TCG TCG GTG GAC TGG ATG AC 774 Deletion of 351 bp in Zygmunt et al. (2009)
BMEI1426-BMEI1427 in B. canis
BMEI1427 ATG GTC CGC AAG GTG CTT TT
BR1080f CCC TTG GTT TGT AGC GGT TG 197 Deletion of 162 bp in BR1080 in Halling et al. (2005)
B. abortus and B. melitensis
BR1080r TCA TCG TCC TCC GTC ATC CT
BMEI1688 TCA ACT GCG TGA ACA ATG CT 278 Deletion of 20,883 bp in Rajashekara et al. (2004),
BMEI1674-BMEI1703 in Halling et al. (2005)
B. suis and B. canis
BMEI1687 GCG GGC TCT ATC TCA AGG TC
BMEI0205f CGT CAA CTC GCT GGC CAA GAG 299,425 Derived from VNTR Bruce 11 Le Flèche et al. (2006)
BMEI0205r GCA GGA GAA CCG CAA CCT AA 551,614
a
Designation are based on the B. melitensis (BME) or B. suis (BR) genome sequences. f, forward; r, reverse.
isolates from different geographic origins and different applied to a total of 99 B. suis field isolates (see
animal species was examined (see supplemental material). supplemental material). All isolates from B. suis biovars
When needed, Brucella reference strains were used. All 1, 2 and 4 displayed the same PCR profiles to the
Brucella isolates were typed according to standard corresponding type strains (Fig. 1), consistent with the
procedures (Alton et al., 1988). Growth and harvesting biovar assigned by biochemical and molecular characteri-
of Brucella cells and bacterial DNA extraction were zation. The only exceptions were few B. suis field isolates
performed as described elsewhere (Garcı́a-Yoldi et al., identified by standard microbiological tests as biovar 3
2006, 2007). (see supplemental material). Two of these isolates (refs.
03-3081-02 and 01-1825-04) were typed as biovar 3 also
2.2. Molecular typing by MLVA but were classified as biovar 1 by PCR-RFLP for
omp genes, AMOS-ery-PCR and the new Suis-ladder. On the
Isolates were subjected to different PCR-based typing other hand, three isolates (refs. 04-3025-03, 04-1361-S4,
techniques: PCR-RFLP for omp2a, omp2b and omp31 genes 04-1361-S5) were typed as biovar 3 only by standard
(Cloeckaert et al., 1995; Vizcaı́no et al., 1997), multiplex microbiological procedures but as biovar 1 by all other
AMOS-ery-PCR (Ocampo-Sosa et al., 2005), original multi- molecular techniques. The existence of B. suis field strains
plex Bruce-ladder (Garcı́a-Yoldi et al., 2006; López-Goñi typed as biovar 3 with the classical biochemical scheme
et al., 2008), and Brucella MLVA (Le Flèche et al., 2006). A that fit with the genetic profiles of biovar 1 has been
new multiplex PCR (Suis-ladder) able to discriminate described by others (Ferrao-Beck et al., 2006; Le Flèche
among all five biovars of B. suis and B. canis was developed. et al., 2006; Whatmore et al., 2006; Fretin et al., 2008;
For this, four pairs of oligonucleotide primers were Huber et al., 2009) and confirms that the taxonomical
designed and selected based on biovar-specific genetic situation of B. suis biovar 3 can be questioned (Fretin et al.,
differences (Table 1). Amplification of DNA was performed 2008).
in 25 ml of a reaction mixture containing 1 ml of template When the Suis-ladder PCR was applied to the B. canis
DNA, 200 mM of each dNTP (Promega Corp.), 2 mM of reference strain a specific PCR profile clearly different from
MgCl2, 1 U of Immolase DNA polymerase and its amplifi- B. suis was obtained: only two fragments of 614 and 197 bp
cation buffer (Bioline Ltd.), and 12.5 pmol of each primer. were amplified (Fig. 1). Interestingly, the 774 bp fragment
After an initial denaturation at 95 8C for 7 min, the PCR common to all B. suis biovars was not obtained with B. canis
profile was set as follows: 35 s of template denaturation at
95 8C, 45 s of primer annealing at 63 8C, and 60 s of primer
extension at 72 8C, for a total of 30 cycles, with a final
extension at 72 8C for 6 min. PCR products were analyzed
by standard 1.5–2.0% agarose electrophoresis.
Acknowledgements
Fig. 2. Bruce-ladder v2.0 multiplex PCR: differentiation of all known Appendix A. Supplementary data
Brucella species and vaccine strains. Lane 1, molecular marker molecular
marker 1 kb plus DNA ladder (Invitrogen Ltd.); lane 2, B. abortus; lane 3, B. Supplementary data associated with this article can be
melitensis; lane 4, B. ovis; lane 5, B. suis; lane 6, B. canis isolate with a B. found, in the online version, at doi:10.1016/j.vet-
canis profile in the original Bruce-ladder; lane 7, B. canis isolates with a B.
mic.2011.06.035.
suis profile in the original Bruce-ladder; lane 8, B. abortus S19; lane 9, B.
abortus RB51; lane 10, B. melitensis Rev.1; lane 11, B. neotomae; lane 12, B.
pinipedialis; lane 13, B. ceti; lane 14, B. microti; lane 15, B. inopinata.
References
Alton, G.G., Jones, L.M., Angus, R.D., Verger, J.M., 1988. Techniques for the
DNA. The Suis-ladder PCR was then applied to a total of 17 Brucellosis Laboratory. Institut National de la Recherche Agronomi-
B. canis field isolates (see supplemental material), included que, Paris.
6 B. canis strains that previously were misidentified as B. Cloeckaert, A., Verger, J.M., Grayon, M., Grépinet, O., 1995. Restriction site
polymorphism of the genes encoding the major 25 kDa and 36 outer-
suis by the original Bruce-ladder PCR. All B. canis field
membrane proteins of Brucella. Microbiology 141, 2111–2121.
isolates gave the same PCR profile to the corresponding B. Ferrao-Beck, L., Cardoso, R., Muñoz, P.M., de Miguel, M.J., Albert, D., Ferreira,
canis type strain, confirming the identity of all isolates as B. A.C., Marı́n, C.M., Thiebaud, M., Jacques, I., Grayon, M., Zygmunt, M.S.,
Garı́n-Bastuji, B., Blasco, J.M., Sa, M.I., 2006. Development of a multi-
canis. The Suis-ladder PCR was also able to differentiate B.
plex PCR assay for polymorphism analysis of Brucella suis biovars
microti strain by the presence of two bands of 774 and causing brucellosis in swine. Vet. Microbiol. 115, 269–277.
197 bp (Fig. 1). Fretin, D., Whatmore, A.M., Al Dahouk, S., Neubauer, H., Garin-Bastuji, B.,
In the original Bruce-ladder protocol PCR seven Albert, D., Van Hessche, M., Ménart, M., Godfroid, J., Walravens, K.,
Wattiau, P., 2008. Brucella suis identification and biovar typing by
amplicons (1682, 1071, 794, 587, 450, 272 and 152 bp) real-time PCR. Vet. Microbiol. 131, 376–385.
were expected from B. suis DNA, and B. canis was Garcı́a-Yoldi, D., Marı́n, C.M., de Miguel, M.J., Muñoz, P.M., Vizmanos, J.L.,
distinguished by the absence of the 794 bp fragment López-Goñi, I., 2006. Multiplex PCR assay for the identification and
differentiation of all Brucella species and the vaccine strains Brucella
(Garcı́a-Yoldi et al., 2006). However, as mentioned before, abortus S19 and RB51 and Brucella melitensis Rev1. Clin. Chem. 52,
some B. canis strains were identified erroneously as B. suis. 779–781.
To overcome this problem, here we also propose the Garcı́a-Yoldi, D., Le Fleche, P., De Miguel, M.J., Muñoz, P.M., Blasco, J.M.,
Cvetnic, Z., Marı́n, C.M., Vergnaud, G., López-Goñi, I., 2007. Compari-
advancement of this PCR protocol by substituting the son of multiple-locus variable-number tandem-repeat analysis with
primers BMEI1436f/BMEI1435r in the original primer mix other PCR-based methods for typing Brucella suis isolates. J. Clin.
by the new primers BMEI1426/1427 (Table 1), which Microbiol. 45, 4070–4072.
Garin-Bastuji, B., Hars, J., 2001. Situation epidemiologique de la brucellose
amplified a 774 bp fragment from the wbkF-wbkD region a Brucella suis biovar 2 en France. Bull. Epidemiol. 2.
(Zygmunt et al., 2009). In addition, the primer pair Halling, S.M., Peterson-Burch, B.D., Bricker, B.J., Zuerner, R.L., Qing, Z., Li,
identifying B. microti described before was also included L.L., Kapur, V., Alt, D.P., Olsen, S.S., 2005. Completion of the genome
sequence of Brucella abortus and comparison to the highly similar
(Mayer-Scholl et al., 2010). An example of the modified
genomes of Brucella melitensis and Brucella suis. J. Bacteriol. 187,
Bruce-ladder PCR (called Bruce-ladder v2.0) is presented in 2715–2726.
Fig. 2. The new primer pair did not interfere with the other Huber, B., Scholz, H.C., Lucero, N., Busse, H.J., 2009. Development of a PCR
primers present in the cocktail mix. All B. canis field strains assay for typing and subtyping of Brucella species. Int. J. Med. Micro-
biol. 299, 563–573.
tested were distinguished to B. suis by the absence of the Le Flèche, P., Jacques, I., Grayon, M., Al Dahouk, S., Bouchon, P., Denoeud,
774 bp fragment, confirming that the deletion of this F., Nockler, K., Neubauer, H., Guilloteau, L.A., Vergnaud, G., 2006.
genetic region is specific for B. canis and can be used in the Evaluation and selection of tandem repeat loci for a Brucella MLVA
typing assay. BMC Microbiol. 6, 9.
Bruce-ladder v2.0 to discriminate between B. suis and B. López-Goñi, I., Garcı́a-Yoldi, D., Marı́n, C.M., de Miguel, M.J., Muñoz, P.M.,
canis. Blasco, J.M., Jacques, I., Grayon, M., Cloeckaert, A., Ferreira, A.C.,
In conclusion, for the differentiation of all B. suis Cardoso, R., Corrêa de Sá, M.I., Walravens, K., Albert, D., Garin-Bastuji,
B., 2008. Evaluation of a multiplex PCR assay (Bruce-ladder) for
biovars, B. canis and B. microti we propose a two steps PCR molecular typing of all Brucella species, including the vaccine strains.
assays. First, by the advancement Bruce-ladder v2.0 PCR it J. Clin. Microbiol. 46, 3484–3487.
is possible to identify correctly all known Brucella species Mayer-Scholl, A., Draeger, A., Göllner, C., Scholz, H.C., Nöckler, K.J., 2010.
Advancement of a multiplex PCR for the differentiation of all cur-
and vaccine strains. Second, by the new Suis-ladder PCR it rently described Brucella species. Microbiol. Methods 80 (1), 112–114.
is possible differentiate the B. suis biovars. These rapid and OIE, 2009. Manual of diagnostic test and vaccines for terrestrial animals.
robust multiplex PCR systems are important tools for In: Bovine Brucellosis, Office International des epizootics, Paris, Chap-
ter 2.4.3.
routine laboratories for effective diagnosis of animal
Ocampo-Sosa, A.A., Agüero-Balbin, J., Garcı́a-Lobo, J.M., 2005. Develop-
brucellosis in domestic and wild animals, and can ment of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9
contribute to the control and eradication of the disease. and the new subgroup 3b of biovar 3. Vet. Microbiol. 110, 41–51.
I. López-Goñi et al. / Veterinary Microbiology 154 (2011) 152–155 155
Rajashekara, G., Glasner, J.D., Glover, D.A., Splitter, G.A., 2004. Compara- a large deletion in Brucella abortus, and other species-specific mar-
tive whole-genome hybridization reveals genomic islands in Brucella kers. Microbiology 143, 2913–2921.
species. J. Bacteriol. 186, 5040–5051. Whatmore, A.M., Shankster, S.J., Perrett, L.L., Murphy, T.J., Brew, S.D.,
Scholz, H.C., Hubalek, Z., Sedlacek, I., Vergnaud, G., Tomaso, H., Al Dahouk, Thirlwall, R.E., Cutler, S.J., MacMillan, A.P., 2006. Identification and
S., Melzer, F., Kämpfer, P., Neubauer, H., Clockaert, A., Maquart, M., characterisation of variable-number tandem-repeat markers for typ-
Zygmunt, M.S., Whatmore, A.M., Falsen, E., Bahn, P., Göllner, C., ing of Brucella spp. J. Clin. Microbiol. 44, 1982–1993.
Pfeffer, M., Huber, B., Busse, H.J., Nöckler, K., 2008. Brucella microti Zygmunt, M.S., Blasco, J.M., Letesson, J.J., Cloeckaert, A., Moriyón, I., 2009.
sp. nov., isolated from the common vole Microtus arvalis. Int. J. Syst. DNA polymorphism analysis of Brucella lipopolysaccharide genes
Evol. Microbiol. 58, 375–382. reveals marked differences in O-polysaccharide biosynthetic genes
Vizcaı́no, N., Verger, J.M., Grayon, M., Zygmunt, M.S., Cloeckaert, A., 1997. between smooth and rough Brucella species and novel species-spe-
DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for cific markers. BMC Microbiol. 13 (9), 92.