Emergence of a Clonal Lineage of Brucella abortus Biovar 3 in Clinical Cases in Spain

Brucella abortus biovar 3 is an uncommon cause of human brucellosis in Spain, where Brucella melitensis has accounted for 97.5% of all cases. B. abortus biovar 3 was detected in specimens from four patients in Cáceres (west-central Spain) with a febrile syndrome associated with arthralgia and myalgia and from one patient in Lugo (northwestern Spain) with orchiepididymitis (March 2004 and April 2005). For tracing, these isolates were studied by biotyping, analysis of 16S rRNA genes, rpoB, gyrA, parC, and housekeeping genes, multilocus variable-number tandem-repeat analysis (MLVA-16), and analysis of hypervariable octameric oligonucleotide fingerprints (HOOF-prints).

Isolates were identified as B. abortus biovar 3 (3) by their CO₂ requirements, H₂S production, agglutination in anti-A-positive and anti-M-negative monospecific sera, and growth on thionin and basic fuchsin (20 μg/ml). They showed a single 16S rRNA gene and rpoB sequences (6, 9). The latter gene harbored substitutions (243-GAC, 268-ACT, and 340-GAA) with respect to the B. melitensis 16 M sequence under GenBank accession no. AE009516 (9). The replacement at codon 268 is thought to be a B. abortus marker because of its presence in reference biovars. The rpoB sequence showed a 100% match with the sequence of B. abortus biovar 7 strain 63/75 (GenBank accession no. DQ086138) (9). The authenticity of B. abortus biovar 7, however, has been questioned for many years (2, 5) because the reference strain was a mixture of B. abortus biovars 3 and 5. Further work showed the obtained rpoB gene differs at position 340 from the sequence of B. abortus biovar 7 reference strain Tulya (GenBank accession no. AY562180) (9). The Tulya strain and other African B. abortus biovar 3 isolates show distinct genetic patterns versus European isolates of the same biovar when analyzed with different genetic markers. The division of B. abortus biovar 3 into two groups has been proposed: 3a, containing the Tulya and African field isolates; and 3b, containing the European isolates (including those we analyzed) (10). Therefore, analyses of our five B. abortus biovar 3 isolates differed from those for the Tulya strain (9).

The gyrA and parC amplifications and sequencing were performed with the primers Bru_gyrA + 4651 (5′-TGCAGCGGTCTTATCTTGATT-3′), Bru_gyrA-5589 (5′-CAAACGAGGTCTGCAAAGG-3′), Bru_parC + 2040 (5′-CAAGCTGACCGAGCTTGAA-3′), and Bru_parC-2871 (5′-CACGAAGGCCGTCAGTATATC-3′) to provide polymorphisms related to the bacterial identification (7). All showed one gyrA (5285-T → C) mutation and one parC [2600-Ala(GCT) → Val(GTT)] mutation compared to B. melitensis 16 M. The gyrA change appears in B. abortus biovars and in Brucella suis, while the parC mutation is seen only in B. abortus biovars. The isolates showed the same sequence type, ST5 (gap-2 aroA-1 glk-1 dnaK-2 gyrB-1 trpE-4 cobQ-1 omp25-1 int-hyp-1) (11), identical to the corresponding sequence of the B. abortus biovar vaccine isolates S19 and RB51. The Spanish isolates differed at two (glk and trpE) and seven (gap, aroA, glk, dnaK, gyrB, trpE, and cobQ) of the nine housekeeping genes from those of the B. abortus biovar 3 5/93 strain and the Tulya strain, respectively (11).

Five MLVA-16 (1) (genetic similarity range, 80% to 95%) and five HOOF-print (4) (35% to 65%) genotypes were obtained. In a comparison of those genotypes to other MLVA-15 genotypes (8), the Spanish cluster split from other European and African B. abortus biovar 3 isolates (similarity index, <40%), suggesting a different clonal lineage (Fig. 1).

FIG. 1.

Dendrogram of clustered MLVA-16 genotypes of the Spanish B. abortus biovar 3 human isolates (see the Brucella 2007 MLVA database [http://mlva.u-psud.fr]), the European B. abortus biovar 3 (3b) isolates, the African B. abortus biovar 3 (3a) Tulya strain, and the B. abortus biovar 1 RB51 and S19 vaccine isolates (8), generated using the categorical coefficient and UPGMA analysis (unweighted-pair group method using arithmetic averages). MLVA-16 information for Bruce09 was not available for these previously studied strains (8).

In summary, we described the first reported cases of B. abortus biovar 3 causing human brucellosis in Spain. MLVA and HOOF-print genotyping revealed a close genetic relationship between the detected emergent isolates; this is further supported by their identical 16S rRNA gene, rpoB, gyrA, parC, and housekeeping gene sequences.

Nucleotide sequence accession numbers.

GenBank has assigned the following accession numbers: for 16S rRNA, EF192470 to EF192474; for gyrA, EF420872; for parC, EF420873; and for rpoB, EU623594.