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Journal of Clinical Microbiology, July 2000, p. 2602-2610, Vol. 38, No. 7
ClinCyte, LLC, San Diego, California
921211; Department of Veterinary
Pathobiology, College of Veterinary Medicine, Texas A&M University,
College Station, Texas 77843-44672;
Department of Veterinary Science, School of Veterinary
Medicine, Louisiana State University, Baton Rouge, Louisiana
708033; Division of Zoonotic Disease
Research, National Animal Disease Center, U.S. Department of
Agriculture, Ames, Iowa 500104;
Department of Genetics and Plant Breeding, University of
Agricultural Sciences, Bangalore, India 5600655;
and Institute of Animal Health and Veterinary Biologicals,
Hebbal, Bangalore, India 5600246
Received 19 October 1999/Returned for modification 29 December
1999/Accepted 13 April 2000
A multiplex amplification and detection platform for the diagnosis
of Mycobacterium bovis and Brucella abortus
infection simultaneously in bovine milk and nasal secretions was
developed. This system (designated the bovine pathogen detection assay
[BPDA]-PCR) consists of duplex amplification of species-specific
targets (a region of the BCSP31K gene of B. abortus and a
repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe
capture hybridization of amplicons for detection. On the basis of
spiking experiments with normal milk, the analytical sensitivity of the
assay was 800 CFU equivalents/ml of milk for B. abortus and
as low as 4 CFU equivalents per ml of milk for M. bovis.
BPDA-PCR was validated with 45 liver samples from lemmings
experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five
culture-negative liver samples as positive (41.7%). Field studies for
the evaluation of BPDA-PCR were performed with samples from dairy
animals from geographically distinct regions (India, Mexico, and
Argentina). A high prevalence of shedding of B. abortus
(samples from India) and M. bovis (samples from Mexico) was
identified by BPDA-PCR. In samples from India, B. abortus
shedding was identified in 86% of milk ring test-positive animals
(n = 15) and 80% of milk ring test-negative cows
(n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools
(n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from
cattle from tuberculin test-positive herds. In contrast, the Argentine
cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of
B. abortus in milk (11.4%). On the basis of these
analyses, we identify BPDA-PCR as an optimal tool for both screening of
herds and testing of individual animals in a disease eradication
program. A combination of the duplex assay, screening of milk samples
in pools, and the proposed algorithm provides a highly sensitive,
cost-effective, and economically viable alternative to serological testing.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Multiplex Approach to Molecular Detection of Brucella
abortus and/or Mycobacterium bovis Infection
in Cattle
*
Corresponding author. Mailing address: ClinCyte, LLC,
11055 Flintkote Ave., STE H, San Diego, CA 92121. Phone: (858)
457-9669. Fax: (858) 457-1827. E-mail:
bookout8{at}earthlink.net.
Journal of Clinical Microbiology, July 2000, p. 2602-2610, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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