Brucellosis 2003 proceedings - PHIDIAS

Poster Session
mutants serves two purposes. First, mutants may be used without concern for the
spread of antibiotic resistance. Second, unmarked, non-polar deletions will confirm
that the previously observed attenuation was due to the interrupted gene and not to
polar effects resulting from transposon insertion. Knockouts were constructed via
overlapping extension PCR and subsequent SacB counter-selection. The mouse
model was used to compare survival of marked (kanamycin resistant-Km R ) to
unmarked deletions (Km S ) and wild-type. Both marked and unmarked deletions
exhibited the reduction in virulence relative to the parental wild-type while no
noticeable difference in attenuation was observed between marked and unmarked
mutants. In addition, mutants made in either B. abortus or B. melitensis revealed no
phenotypic differences in the mouse model resulting from differences in genetic
background. Several of the mutants have been evaluated in the pregnant goat
model, in order to compare safety. One mutant, BMV2, caused neither
seroconversion nor abortion in the goats, which was predicted by rapid clearance in
the mouse model. A non-rapid clearing mutant, BM8, colonized the dams but was
unable to colonize the fetuses. Future plans include evaluating the efficacy of these
mutants including several correlates of protective immunity.
94- NEW SYSTEM TO ENCAPSULATE ANTIGENIC EXTRACTS FROM Brucella
ovis FOR VACCINAL PURPOSES.
M. Estevan 1 , C. Gamazo 1 , M. J. Grilló 2 , J. M. Blasco 2 , C. M. Marín 2 , G. García del Barrio 3 and J. M.
Irache 3 . (1) Departamento de Microbiología, Universidad de Navarra, Pamplona, Spain. (2) Unidad de
Sanidad Animal, SIA-DGA, Zaragoza, Spain. (3) Centro Galénico, Universidad de Navarra, Pamplona,
Spain.
Vaccination against Brucella infections in animals is usually performed by
administration of live attenuated smooth Brucella strains. However, live vaccines
present some inconvenient, such as the residual virulence or the interference with
serological diagnosis. Therefore, it was previously developed an innocuous
subcellular vaccine based on microparticles (Mp) as transport vehicles (adjuvants) for
the control release of antigenic extract of Brucella ovis (HS) [Murillo et al., Vaccine 19
(2001): 4099-4106]. Founded on that approach, we have now designed a procedure
to prepare microparticles by a w/o/w emulsion solvent evaporation method carried
out by TROMS (Total Recirculation One-Machine System). This method is a new
procedure based on the formation of a multiple emulsion by the injection of the
phases under a turbulent regime, avoiding the use of aggressive homogenisation
techniques. The method is easily reproducible and applicable on a semi-industrial
scale, permits complete control of all production parameters and can be performed
under sterile conditions [García de Barrio et al., J. Contr. Rel. 86 (2003) 123-130]. Mp
characterization was performed according to the following parameters: size (2.09 ±
0.85 µm), zeta potential (-20.05 ± 0.90 mV), HS content (15.2 ± 1.9 µg/mg),
encapsulation efficiency (73.5 ± 13.7 %), distribution of HS on/in microparticles
(surface 8.4%, strongly binding to surface 11.1 %, inside of particle 80.5 %). Different
parameters may affect on the characteristics of resulting Mp (such as pharmaceutical
auxiliaries and cyclodextrins). Thus, around 32 % of the loaded HS was released
from the formulations without β-cyclodextrin (β-CD) and Pluronic® F-68 (F 68). In
contrast, the release of HS from formulations without with both components was only
4.2 %, and 15.5 % when F-68 was omitted. Chemical and serological analysis
Brucellosis 2003 International Research Conference
143