Brucellosis 2003 proceedings - PHIDIAS

Poster Session
33- IDENTIFICATION OF SMOOTH AND ROUGH FORMS IN CULTURES OF
Brucella melitensis STRAINS BY FLOW CYTOMETRY.
C. M. Fernandez-Prada, E. B. Zelazowska, A. K. Bhattacharjee, M. P. Nikolich, and D. L. Hoover.
Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD 20910. USA.
We developed a flow cytometric method to determine the proportion of B.
melitensis cells displaying surface O-polysaccharide (OPS) in liquid culture. OPS was
detected using polyclonal antibodies from rabbits immunized with smooth (S) or
rough (R) Brucella LPS. First, we evaluated the binding of these antibodies to 16M
(S), WRR51 (R) and complemented WRR51 expressing the wboA gene (S) as well
as to their corresponding GFP-expressing derivative strains 16M/GFP, WRR51/GFP
and WRR51/GFP+wboA. The rough mutants did not react with anti-S-LPS nor did the
smooth strains react with anti-R-LPS. Second, using different ratios of 16M/GFP and
WRR51/GFP, we were able to detect the presence of 1% or fewer rough bacteria
spiked into a sample of smooth organisms. Third, we evaluated the purity of cultures
of B. melitensis strains grown in a fermenter. Finally, we sequentially examined the
spontaneous loss of green fluorescence and surface OPS in stationary phase
cultures of smooth B. melitensis strains 16M/GFP and WRRP1pGSG5. Plasmid
pGSG5, which contains both wboA and GFP genes, complements wboA and
restores a smooth phenotype to rough strain WRRP1 (∆purE∆wboA B. melitensis).
We found that 16M/GFP lost the plasmid faster but displayed OPS longer than
WRRP1pGSG5. After 40 days in culture, only 1% of the population of 16M/GFP was
green. However, more than 70% of 16M/GFP bound anti-S-LPS antibody while 30%
bound anti-R-LPS antibody. In contrast, 5-10% of WRRP1pGSG5 cultured for the
same period of time were still green and bound anti-S-LPS antibody, while at least
80% bound anti-R-LPS antibody. These flow cytometric methods may be useful for
quality control of process development for large-scale vaccine production.
34- EVALUATION OF A RAPID LATERAL FLOW TEST FOR DETECTION OF IgM
AND IgG IN HUMAN BRUCELLOSIS.
J. Douglas 1 , L. Okuhara 1 , T. Abdoel 2 , and H. Smits 2 . (1) Department of Microbiology, University of
Hawaii, Honolulu, Hawaii, USA. (2) KIT Biomedical Research, Royal Tropical Institute, Amsterdam,
The Netherlands.
In 1999, we participated in evaluating a rapid IgM dip stick method for
diagnosis of acute brucellosis and found this three hour test to be both highly
sensitive and specific (J. Clin. Microbiol. 37:4179-4182). Here we describe our results
based on lateral flow technology. Sera were examined from individuals infected with
Brucella suis. These sera had been tested by the dipstick method for IgM. Thirty-four
sera from slaughter house patients were tested. Of the 34 sera 20 were from acute
cases. These sera were positive, ranging from 2+ to 4+ and the results were
comparable with earlier IgM dipstick tests. We also tested 14 sera from treated
patients. Here we found 10 of 14 sera ranged from weakly positive to 2+. One of two
2+ individuals was positive after 24 months since diagnosis, suggesting a relapse or
treatment failure. The other was 2+ after one month of treatment. The four remaining
sera from treated individuals were negative. The study included 12 sets of paired
sera (acute and treated). All paired sera showed a decline in the IgM. The IgG lateral
flow test gave varied results ranging from negative to 2+. Thirteen of the 20 acute
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