A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle

Citation
S. Sreevatsan et al., A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle, J CLIN MICR, 38(7), 2000, pp. 2602-2610
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Clinical Immunolgy & Infectious Disease",Microbiology
Journal title
JOURNAL OF CLINICAL MICROBIOLOGY
ISSN journal
0095-1137 → ACNP
Volume
38
Issue
7
Year of publication
2000
Pages
2602 - 2610
Database
ISI
SICI code
0095-1137(200007)38:7<2602:AMATMD>2.0.ZU;2-R
Abstract
A multiplex amplification and detection platform for the diagnosis of Mycob acterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pat hogen detection assay [BPDA]-PCR) consists of duplex amplification of speci es-specific targets (a region of the BCSP31K gene of B. abortus and a repea t-sequence region in the hsp65 gene of nl. bovis, respectively). This is fo llowed by a solid-phase probe capture hybridization of amplicons for detect ion. On the basis of spiking experiments,vith normal milk, the analytical s ensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus a nd as low as 4 CFU equivalents per mi of milk for dl. bovis. BPDA-PCR was v alidated,vith 45 liver samples from lemmings experimentally infected with B . abortus. The assay sensitivity, based on culture status as a "gold standa rd," was 93.9%. In this experiment, BPDA-PCR also identified five culture-n egative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed,vith samples from dairy animals from geographic ally distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and dl. bovis (samples from Mex ico) 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 w as 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 fro m 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 mi lk (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 t esting of individual animals in a disease eradication program. A combinatio n of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically vi able alternative to serological testing.