Brucellosis is a widespread zoonosis of great economic importance caused by facultative intracellular Gram-negative bacteria belonging to the genus Brucella. Although brucellosis in domestic animals has been eradicated in great number of European countries, the risk of reintroduction of the disease still exists through spill-over from wildlife that are considered to be natural reservoirs . A study on the surveillance of different swine pathogens demonstrated the presence of Brucella suis biovar 2 in a population of wild boars in Switzerland [2, 3].
Reliable and sensitive diagnostic tools play a crucial role in the control of brucellosis in livestock, wildlife and humans. Although blood and tissue cultures remain the 'gold standard' for diagnosis, they show low sensitivity, are time consuming, and represent a risk for laboratory personnel [4, 5].
Serology is a standard method for the epidemiological surveillance of brucellosis [2, 3, 6–9]. However, cross-reactions between Brucella species and other Gram-negative bacteria, such as Yersinia enterocolitica O:9, Francisella tularensis, Escherichia coli O:157, Salmonella urbana group N, Vibrio cholerae and Stenotrophomonas maltophilia, are a major problem of the serological assays [10–13]. The source of antigenic cross-reactions is the O-chain of the smooth lipopolysaccharide (S-LPS) present on the surface of the bacterial cell, which shows great similarity in smooth Brucella spp. and the abovementioned bacteria . False-positive serological results due only to Y. enterocolitica O:9 affect up to 15% of the cattle herds in regions free from brucellosis, generating considerable additional costs for surveillance programs . False-negative results have also been observed in serological diagnosis of brucellosis [11, 15–17]. They occur mostly due to the fact that the antibody response is dependent upon the stage of infection during sample collection . For example, Leal-Klevezas and colleagues stated that detectable amounts of antibodies are not recorded in the first 12–16 days after artificial inoculation of goats with Brucella abortus . On the other hand, when the disease becomes chronic, the antibody titre could fall to undetectable levels [17, 20], which is especially the case with intracellular organisms like Brucella spp. . Latent infection without seroconversion further complicates the problem, particularly in pre-pubertal animals .
Molecular diagnostic techniques represent an important breakthrough in the diagnostic practice. A number of genus- or species-specific conventional PCR assays using primers derived from different gene sequences from the Brucella genome, such as 16S rRNA , the 16S-23S intergenic spacer region , omp2  and bcsp31 , have been established. These assays were adapted for application to Brucella detection in different clinical specimens. In the majority of studies, conventional PCR proved to be a good means to detect Brucella DNA from clinical specimens [27–35], while Romero and colleagues found that PCR had lower sensitivity compared to the conventional detection methods .
The introduction of real-time PCR offers improved sensitivity, specificity and speed of performance compared with conventional PCR. Several real-time PCR assays using different detection chemistries have already been established for Brucella identification [37–39]. Moreover, some of them were evaluated with various clinical samples of human and animal origins [40–45]. Most of the authors confirmed that real-time PCR was a very sensitive method of detection from clinical samples [41, 43, 44]; nevertheless, O'Leary and colleagues found that there was no advantage in using real-time PCR on blood, milk and lymph node samples of naturally infected cows over standard serological and bacteriological methods .
The goal of our present study was to evaluate the performance of a recently described real-time PCR assay  for Brucella spp. detection with wild boar blood and tissue samples collected under the wild boar surveillance program in Switzerland. This assay is based on the Brucella spp. specific multiple IS711 insertion sequence and therefore shows great sensitivity . The same samples were additionally tested by bacterial isolation and three serological tests (i-ELISA, c-ELISA and RBT), and the results obtained were compared to those of the real-time PCR.