Brucellosis 2003 proceedings - PHIDIAS

Poster Session
constituents that are immunogenic or antigenic for humans. Outer membrane
proteins are particlularly attractive for this purpose. In the present study, we cloned,
expressed and purified five predicted outer membrane proteins of B. suis, including
two large ones of 88 kD and 68 kD molecular weight. The recombinant proteins were
designed with 6-his and V5 epitope tags at their C termini to facilitate detection and
purification. The B. suis genes were PCR synthesized based on their ORF
sequences and directly cloned into an entry vector. The recombinant entry constructs
were propagated in TOPO 10 cells, recombined into a destination vector, pET-
DEST42, then transformed into E. coli BL21 cells for IPTG-induced protein
expression. The expressed recombinant proteins were confirmed with western blot
analysis using anti-6-his antibody conjugated with alkaline phosphatase. These B.
suis outer membrane proteins were purified by immuno-capture using a specific
antibody against the V5 epitope. Spleen cells from mice previously immunized with
the purine auxotrophic live, attenuated vaccine strain B. melitensis WR201 were
cultured with recombinant proteins. After 24 hours, culture supernatant fluids were
assayed for IFNγ and interlukin 2 content. Recombinant B. suis proteins were readily
expressed and purified by this approach and stimulated cytokine production from B.
melitensis-immune mouse spleen cells.
83- HUMORAL AND CELLULAR IMMUNE RESPONSE TO Brucella abortus
STRAINS RB51 AND S19 IN HEREFORD HEIFERS IN PATAGONIA REGION,
ARGENTINA.
Robles, C.A. 1 , Abalos, P. 2 , Cabrera, R. 1 , Petray, S. 1 , Ibarra, L. 2 . (1) National Institute for Agricultural
Technology– CC: 277 (8400) Bariloche, Argentina. (2) Facultad de Ciencias Veterinarias y Pecuarias.
Universidad de Chile - Casilla 2, Correo 15, Santiago. Chile.
A controlled field study was carried out using two homogeneous groups of 25
nine-month-old Hereford heifers. On day cero all the animals were bled and
subcutaneously vaccinated with commercially S19 or RB51 vaccines at
recommended doses. Both groups were bled at days 30, 90, 210 and 360 post
vaccination. On each opportunity blood with anticoagulant was taken and cultivated
in sterile plates by duplicates. One was incubated adding a cytosolic antigen of
Brucella abortus RB51 while the other with PBS as control. After 18 hours of
incubation at 37ºC with an atmosphere of 5% of CO 2 , plasma was obtained. Also
blood samples without any anticoagulant were taken for sera collection. The sera
samples were processed with BPAT and with two standard I-ELISA, using S-LPS (for
S19 antibodies) or R-LPS (for RB51 antibodies) respectively. The plasma samples
were processed with a capture ELISA (Bovigamtm, CSL), for the detection of bovine
γ-interferon (INFγ). The humoral immune response generated by RB51 measured
with an I-ELISA using a R-LPS was similar to the one generated by S19, measured
with an I-ELISA using a S-SPLS. In both cases, on day 30, similar antibody picks
were produced with the following figures: pp 90.4 ± 26.6 for RB51 vs. pp 91.8 ± 21
for S19. RB51 vaccine showed a cellular immune response, measured through the
production of INFγ, similar to the one produced by S19 (pp 22.1 ± 11.6 for RB51 vs.
pp 20.5 ± 14.7 for S19). Brucella abortus RB51 generated, in comparison to S19, a
similar humoral and cellular immune response without any interference with
diagnosis. It could be a good alternative for adult animal vaccination in areas were it
136
Brucellosis 2003 International Research Conference