Brucellosis 2003 proceedings - PHIDIAS

Poster Session
was comparable to these previously described PCR protocols. The assay showed a
high reproducibility of 96 % to 99 % and was lineal in a range between 101 and 108
fg of Brucella DNA. The coefficient of regression of the standard curve was on
average 0.98. The intra-assay and the inter-assay coefficient of variation of the
threshold cycle were 1.3% and 6.63% respectively. For accurate quantification of the
number of copies of Brucella in samples containing unknown quantities, we have
used serial dilutions of a vaccine B. abortus B-19. The fact that no-signal were
obtained with closely related organisms argues for a high specificity of this newly
developed method.
In conclusion, the high sensitivity, simplicity and reproducibility of the real-time
Brucellosis DNA quantification method, makes it especially suitable for Brucellosis
diagnosis, as capillaries do not have to be reopened for post-PCR analysis, the risks
of carry-over contamination could be minimised.
43- REAL-TIME PCR ASSAY FOR FIELD DIAGNOSIS OF Brucella abortus IN
WILDLIFE POPULATIONS IN YELLOWSTONE NATIONAL PARK.
D.T. Newby and F.F. Roberto. Biotechnology Department, Idaho National Engineering and
Environmental Laboratory, Idaho Falls, Idaho, USA 83415-2203.
Brucellosis is estimated to be one of the top five zoonoses globally, with
outbreaks remaining common in both wildlife and domesticated animals. Bovine
brucellosis has not yet been eradicated in the U.S., and wildlife species, such as
bison and elk, remain an important potential reservoir for the disease. We have
developed a real-time PCR assay for B. abortus using a field-portable instrument for
use in diagnosing infection in wildlife to facilitate improved wildlife management, and
thus minimize the potential for transmission of the disease to cattle that range near
Yellowstone National Park.
Blood, tissue, and amniotic fluid have been screened using this assay which
targets a 156 bp amplicon encompassing portions of the alkB gene and the IS711
insertion element of B. abortus. Using genomic DNA as template, this assay is linear
over 7 orders of magnitude (7.5 ng down to 7.5 fg). This lower detection limit
corresponds to 2 genomic copies; in tissues the lower detection limit increases to 50
to 500 genomic copies. Concentrations of B. abortus DNA in cow blood samples
ranged from less than 7.5 fg (estimated to be 2.1 x 10 4 cells/ml blood) to as high as
1.8 pg (estimated to be 5.0 x 10 6 cells/ml blood) in a sample from a spontaneous
abortion. A similar infectious load was estimated in the amniotic fluid of an infected
bison cow (6.8 x 10 6 cells/ml), while analysis of secondary sex organ tissue from an
infected bison indicated a much lower load of 4.4 x 10 3 cells/ml. Preliminary real-time
PCR results for blood, tissues, and bodily fluids are in agreement with culture results,
suggesting that this approach may be very useful for field-testing of animals
suspected to be infected with B. abortus.
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