Use of viable bacteria detection to assess long term efficacy of Brucella abortus RB51 vaccination in pregnant water buffaloes expose to challenge

The RB51 vaccine is a live attenuated rough mutant derived from the virulent Brucella abortus 2308 strain. As a DIVA-like vaccine, RB51 allows discrimination between vaccinated and naturally infected animals, thereby facilitating the concurrent application of vaccination strategies alongside test-and-slaughter programs (1). The study was designed to assess the longterm efficacy of the RB51 vaccination protocol authorized in Italy and applied to young (6-9 months) buffaloes (Italian Ministry of Health Authorization n° 524/2024-PR) (Research was funded by Italian Ministry of Health project grant IZSAM 09/19 RC and IZSAM 04/22 RC). The protocol followed the WOAH Manual, with modifications including the administration of a triple vaccine dose followed by a booster 30 days apart (1,2). 10 vaccinated and 6 unvaccinated pregnant water buffaloes were included (3). Following experimental infection with B. abortus biovar 1 (10⁸ CFU), both dams and their aborted foetuses (foetal membranes, placenta, cotyledons, amniotic fluid, yolk sac, and abomasal fluid) or live born calves were examined. Bacterial load was assessed to determine the presence and distribution of viable bacteria. Isolation and enumeration were conducted on different organs (brain, spleen, lung, abomasum), lymph nodes, foetal tissues. Over a two-year period, 103 samples were analyzed: 20 foetal, 54 lymph node, and 29 organ samples. Three additional animals are pending slaughter. Approximately 82% of samples tested negative (47.57% vaccinated, 33.98% unvaccinated), while 18.47% tested positive (17.48% unvaccinated, 0.97% vaccinated) as shown in Figure 1. All unvaccinated animals yielded at least one positive sample. Furthermore, 36.84% of positive cases originated from foetuses, 21.05% from lymph nodes, and 42.11% from organs as shown in Figure 2. Among positive samples, 73.68% showed bacterial loads exceeding 100,000 CFU/g, 10.53% around 1,000 CFU/g, and 15.79% around 100 CFU/g (figure 3). Notably, vaccinated animals exhibited no clinical signs of disease, apart from measurable immunological responses. In the single vaccinated case with bacterial detection, B. abortus biovar 1 was confined exclusively to the retropharyngeal lymph nodes, suggesting effective containment of infection near the primary site of entry. RB51 vaccination in water buffaloes markedly reduces bacterial burden and systemic dissemination, thereby supporting its effectiveness as a key component of integrated brucellosis control and eradication programs.