(Evaluation of) Humoral and cellular immune response evaluation against Brucella abortus strain RB51 antigens in bovine

In order to comparatively evaluate the type of immune response induced by p urified structural Brucella abortus antigens as well as live vaccines, 14 c riollo heifers, 19 months old, were randomly distributed in five experiment al groups and immunised subcutaneously with: B. abortus purified outer memb rane proteins (OMP-II), B. abortus OMP-II coupled to O-chain, viable B. abo rtus strain RB51, viable B, abortus strain 19 (C19). A sterile saline solut ion was used for the control group. Two months after vaccination the animal s were challenged intramuscularly with reference virulent strain B. abortus 2308. 30 days after challenge the protection level was evaluated. The humo ral immune response was evaluated using conventional Rose Bengal agglutinat ion test, complement fixation test, radial immunodiffusion as well as ELISA , western blot and dot blot assays at days 0, 8, 15, 30, 60 and 90. Additio nally, the specific IgG2 isotype response was determined by double sandwich ELISA. To evaluate cellular immune responses, lymphocyte proliferation was measured by timidine incorporation and expressed as stimulation index (S.I .), IFN-gamma activity by ELISA and CD4/CD8 ratio by fluorocitometry. Anima ls vaccinated with live strains presented 100% protection against challenge , while a 66% protection was observed in those vaccinated with purified ant igens. The diagnostic advantage of B. abortus Strain RB51 was evidenced by the lack of humoral immune responses against sLPS in all vaccinated groups except for the strain 19 group. No significant differences (p>0.05) were de tected in any of the groups by lymphoproliferation when stimulated in vitro with purified OMP-II, O-chain or sLPS. When crude soluble RB51 protein was used, S.I. turned significant (p<0.05) and indicated a better response ind uced by the live vaccines. IFN-gamma-ELISA tests with stimulated tissue cul tures performed better when measured 72 hour after antigen exposure. In ref erence to the experimental groups, higher levels were detected in the group s immunised with live vaccine, mainly strain 19. When CD4/CD8 ratio were co nsidered the values were constant during the observation and no differences were observed. The results confirm that B, abortus strain RB51 induced lev el of protection similar to strain 19, and had the advantage of differentia l diagnosis. Purified antigens OMP-II and OMP-II-O-chain, induced a lower l evel of protection but they performed similar to replicating vaccines indic ating the immunodominance of OMP-II, but suggesting the need of multiple do ses to reach the same level of protection. It is proposed that the protecti on observed in animals vaccinated with B. abortus RB51 is the result of sti mulation of both T CD4 Th1 and CD8 lymphocytes by the peptides of the outer membrane proteins.