Lead researcher: 
P. Mª Muñoz Álvaro
96.800 €

The brucellae cause brucellosis, a zoonosis affecting livestock and humans. B. abortus and B. melitensis infect ruminants, and B. suis swine and wildlife. Moreover, sheep are infected by B. avis, a rough (R) species. Vaccination is essential to control brucellosis but the only vaccines useful are attenuated strains (B. melitensis Rev1 in small ruminants, and B. abortus 819 in cattle) that induce false positive reactions in serological tests hampering differentiation of infected and vaccinated animals (DIVA problem).

Moreover, they are not completely safe or stable, and 819 does not induce optimal protection. Rev1 is the only vaccine against B. avis, and when discontinued after B. melitensis eradication, becomes an emerging problem in several EU regions. Porcine brucellosis by B. suis biovar 2 is also an emerging problem in the EU, particularly in outdoor breeding systems because wild boars are infected by biovar 2. There is no vaccine against B. suis and, as infections by cross-reacting bacteria occur frequently in pigs, false positive serological reactions (FP8R) are common. B. avis tests also lack specificity and sensitivity. Thus, new and better vaccines solving the DIVA problem (DIVA vaccines) and better diagnostic tools are necessary.

To solve these problems, the teams in this proposal have developed DIVA methods/patents, and tools to stabilize the LP8 and to improve Brucella vaccines based on the identification of structural (LP8, lipids and outer membrane proteins [Omp]) and metabolic virulence requirements, as well as immunological, bacteriological and molecular diagnostic tools. Several developments concerning B. melitensis and B. abortus (new B. melitensis vaccines, S19 tagged vaccines and PCR typing tools) have been either marketed or taken up for further development and/or application by interested parties and companies. Thus, the present project is aimed to exploit previous findings focusing on the needs identified on B. avis and B. suis brucellosis.

Accordingly, the Objectives are:

  1. to refine the previously obtained DIVA B. avis vaccine candidates and to test them for efficacy and safety in sheep against B. avis under controlled conditions; and
  2. to apply knowledge gained through previous projects to obtain final DIVA B. suis vaccine candidates.
  3. to upgrade the direct and indirect diagnostic tests necessary to complement the DIVA approach, to facilitate the diagnosis of porcine brucellosis in the context of FPSR, and to improve B. avis tests.

Both on B. avis and B. suis biovar 2 backgrounds, DIVA methods will exploit the Green Fluorescent Protein and N-acetyi-0-PS tagging methods developed by the teams involved. Attenuation and improved immunogenicity on both backgrounds will be achieved on the bases of previous findings on Omp-deletion, LPS-core structure disruption (both on 0-PS carrying and 0-PS lacking [i.e. R] mutants). Moreover, for B. suis biovar 2, an extension of previous studies on envelope lipids and metabolism will warrant that a properly balance of immunogenicity/attenuation is obtained, and that a definite B. suis vaccine prototype can be proposed.

Finally, we will carry out a proteomic study of Brucella antigens to identify candidates for circumventing the FPSR problem, and upgrade a partially defined enrichment medium that, combined with the appropriate PCR, could facilitate the diagnosis, including the identification of the new vaccines

Ministerio de Ciencia e Innovación
Project members: 
J. Mª Blasco y Mª Jesús de Miguel y M. Barberán Pelegrín (U. de Zaragoza)