A Multiplex Approach to Molecular Detection ofBrucella abortusand/orMycobacterium bovisInfection in Cattle

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Abstract
A multiplex amplification and detection platform for the diagnosis ofMycobacterium bovisandBrucella abortusinfection 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 ofB. abortusand a repeat-sequence region in thehsp65gene ofM. 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 forB. abortusand as low as 4 CFU equivalents per ml of milk forM. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected withB. 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 ofB. abortus(samples from India) andM. bovis(samples from Mexico) was identified by BPDA-PCR. In samples from India,B. abortusshedding 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. boviswas 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 ofM. bovisshedding in nasal swabs (2.9%) and milk (1.4%) and ofB. abortusin 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.