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United States Department of Agriculture

Agricultural Research Service

Title: Molecular Detection of Brucella Abortus And/or Mycobacterium Bovis Infection in Cattle: a Multiplex Approach

Authors
item Sreevatsan, Srinand - CLINCYTE, SAN DIEGO, CA
item Bookout, Jack - CLINCYTE, SAN DIEGO, CA
item Ringpis, Fidel - CLINCYTE, SAN DIEGO, CA
item Perumaala, Veera - TX A&M, COLLEGE STATION
item Ficht, Thomas - TX A&M, COLLEGE STATION
item Adams, L - TX A&M, COLLEGE STATION
item Hagius, Sue - LA ST UNIV, BATON ROUGE
item Elzer, Philip - LA ST UNIV, BATON ROUGE
item Cooksey, Robert - CDC, ATLANTA, GA
item Bricker, Betsy

Submitted to: Molecular Diagnosis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 13, 2000
Publication Date: N/A

Interpretive Summary: Bovine brucellosis and bovine tuberculosis are major diseases of cattle. The bacteria that are responsible for these infections in cattle can also cause serious disease in humans. Although both diseases are under control in the USA, they still cause serious problems in other countries, especially developing countries. Additionally, the two disease agents can also infect wildlife, which, in turn, can reinfect cattle. For these reasons, rapid and inexpensive tests for bovine brucellosis and bovine tuberculosis are important to agriculture. This paper reports the development of a new test that is rapid and inexpensive. One advantage of this new assay is that it checks for both bovine brucellosis and bovine tuberculosis in a single test. This is valuable since both diseases are often found in the same areas. Performance of the test is relatively easy. Testing can be done in the field by collecting samples of milk or nasal secretions for later evaluation. We hope that this new test will help in controlling the spread of these two important diseases.

Technical Abstract: A multiplex amplification and detection platform to diagnose Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system [designated Bovine Pathogen Detection Assay (BPDA-PCR)] consists of duplex amplification of species-spe targets (a gene region of the 31-kDa OMP of B. abortus and a repeat sequence region in the hsp65 of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. Based on normal milk spiking experiments, the analytical sensitivity of the assay was 40-100 cfu/reaction for B. abortus and as low as 4 cfu for M. bovis. BPDA-PCR was validated on 45 lemming liver samples experimentally infected with B. abortus. The assay sensitivity based on culture status as a "gold standard" was 93.9%. In this experiment, BPDA-PC ntified 5 infected culture negative liver samples as positive (41.7%), thus proving to be more reliable than culture to diagnose brucellosis. Field studies to evaluate BPDA-PCR were performed on dairy animals from geographically distinct regions (India, Mexico and Argentina). Based on these analyses we identify BPDA-PCR as an optimal tool for both herd screening and individual animal testing 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.

Last Modified: 7/22/2014
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